prompt_front: Now, answer the following question based on the text below, only give me the answer and do not output any other words.

Question: When did the London Paving and Lighting Act pass, which mandated the numbering of houses?

Read the following text and answer briefly.

Context 1: A Brief History of Benjamin Franklin's Residences on Craven Street, London: 1757 - 1775 - Journal of the American Revolution
Benjamin Franklin House, 36 Craven St, London  (Photo by Elliott Brown | Wikimedia Commons)
If one looked into Benjamin Franklin’s time on Craven Street, they might initially believe he lived at 36 Craven Street the entirety of his two stays in London based on the plethora of articles on the internet that say so  If they dug a little deeper they might read that he lived at No  27 Craven Street, previously numbered 7, but now numbered 36; or that he lived exclusively at No  7 Craven Street; or that he lived in multiple residences on Craven Street; or that he moved out of No  36 to another house on Craven Street and then moved back into No  36 the last year of his residence  What is one to believe with all of the conflicting accounts? What does the historical record have to say about Franklin’s time on Craven Street?
Figure 1  Spur Alley 1685  “A map of the parish of St Martins in the Fields, taken from ye last survey, with additions (1685)”  (© The British Library Board, Shelfmark: Maps Crace Port  13 2, Item number: 2)
Before Craven Street existed there was Spur Alley, a narrow passageway sandwiched between the Hungerford Market to the north (now Charing Cross Station) and Scotland Yard and the Northumberland House and Garden to the south  It was flanked on both ends by major thoroughfares, the Strand on the west, connecting Westminster to London by road, and the River Thames on the east, not only connecting the two cities to each other and to Southwark on the south side of the Thames, but connecting the entire metropolis to the rest of the world  Being located in the City of Westminster, Spur Alley had escaped the devastation of the Great Fire of London in 1666 leaving its wooden structures, built in the early part of seventeenth century, intact, but also in dire need of restoration or demolition  “The ratebooks show that during the last thirty years or so of their existence the houses in Spur Alley were in a very bad condition  Few of them were rated at more than a few shillings and many of them were unoccupied ”[1] The landowner, William, 5th Baron Craven, desiring to increase the profitability of his assets, tore down the derelict structures on Spur Alley around 1730 and leased the newly established lots to builders  By 1735, twenty brick houses in the Georgian style had been built on the west side and sixteen on the east side of the way now called Craven Street [2]
Figure 2  Craven Street 1746  (John Rocque London, Westminster and Southwark, First Edition 1746, Motco Enterprises Limited, motco com)
Letters to Franklin during his residence with Mrs  Margaret Stevenson, his landlady on Craven Street, were addressed rather vaguely; “Craven Street/Strand”, “Mrs  Stevensons in Craven Street”, or “Benjamin Franklin Esqr ” are but a few examples  Letters from Franklin referenced “London,” or sometimes “Cravenstreet,” but never included a number  Despite the absence of numbered addresses in Franklin’s correspondence, there was a sense of one’s place in the neighborhood based on entries in the Westminster Rate Books (tax assessments)  The Rate Books did not list house numbers during Franklin’s time there, but they did list the residents of Craven Street in a particular order that became the default numbering system for the street  Number one was associated with the first resident listed under “Craven Street” in the Rate Books and was the northernmost house on the west side of the street  The numbers increased counter-clockwise down the west side and up the east side in accordance with the list of residents  In 1748, the first year of Margaret Stevenson’s (Stevens in the Rate Books for that year) residence on Craven Street, she is listed as the twenty-seventh resident, the second house north of Court Street (later Craven Court, now Craven Passage) on the east side of the street [3]
In 1766, Parliament passed the London Paving and Lighting Act (6 Geo  3 c  26), “An act for the better paving, cleansing, and enlightening, the city of London, and the liberties thereof; and for preventing obstructions and annoyances within the same; and for other purposes therein mentioned ”[4] One of the other purposes therein mentioned was the numbering of houses  With an aim to bring order to the chaotic numbering systems or lack thereof on London streets the Act provided that “… the said commissioners … may also cause every house, shop, or warehouse, in each of the said streets, lanes, squares, yards, courts, alleys, passages, and places, to be marked or numbered, in such manner as they shall judge most proper for distinguishing the same ”[5] This was quite an undertaking that took years to accomplish  It was a decade later before numbered addresses on Craven Street in the City of Westminster appeared in The London Directory (1776)  The London Directory and its competitors were published primarily by booksellers or printers to supplement their income and were highly profitable  To say they were competitive is an understatement  “Some of the most hotly disputed struggles over copyright in the century concerned guidebooks  Many were optimistically emblazoned with a royal license and a notice that the work had been entered at Stationers’ Hall  Various struggles between rival guides intensified as the potential for profits became clear ”[6] The London Directory boldly proclaimed to contain “An ALPHABETICAL LIST OF THE NAMES and PLACES of ABODE of the MERCHANTS and PRINCIPAL TRADERS of the Cities of LONDON and WESTMINSTER, the Borough of SOUTHWARK, and their Environs, with the Number affixed to each House ”[7] Kent’s Directory made a similar proclamation: “An Alphabetical LIST OF THE Names and Places of Abode OF THE DIRECTORS of COMPANIES, Persons in Public Business, MERCHANTS, and other eminent TRADERS in the Cities of London and Westminster, and Borough of Southwark WITH THE NUMBERS as they are affixed to their Houses agreeable to the late Acts of Parliament ”[8] Mrs  Stevenson wasn’t included in the directories because she didn’t meet the criteria of being a merchant or trader, not because she was a woman  Although it is rare to see women listed in the directories, some examples do exist [9] If Mrs  Stevenson had appeared in the directories in 1776 it would not have been on Craven Street as she had moved to Northumberland Court, a stone’s throw away, the previous year [10] A comparison of Craven Street residents whose names and addresses do appear in the directories with the same residents as they appear in the Westminster Rate Books determines if the numbering systems were congruent  For the most part they were  For example, Joseph Bond at No  30, William Rowles at No  31, Samuel Sneyd at No  32, and Jonathan Michie at No  35 in The London Directory coincide with their places of residence in the Westminster Rate Books; however, errors did occur  The 1776 edition of The London Directory lists Brown & Whiteford, wine merchants, at No  9 Craven Street while the Westminster Rate Books list them as the twenty-ninth residents  Obviously, it makes no sense to have Brown & Whiteford at No  9 in The London Directory and their next-door neighbor, Joseph Bond, at No  30  The same error appears in Baldwin’s The New Complete Guide for 1783  The New Complete Guide may have “borrowed” the error from The London Directory  It was not uncommon for the owner of one directory to copy entries from another to save both time and money  Beginning in 1778 and contrary to The London Directory, Kent’s Directory faithfully followed the numbering system of the Westminster Rate Books in all of its editions and listed Brown & Whiteford at No  29 as did Bailey’s Northern Directory in 1781  Perhaps realizing their error, The London Directory changed their listing of Brown & Whiteford from No  9 to No  29 in their 1783 edition and maintained that listing thereafter 
Sometime prior to 1792, the embankment on the Thames at the south end of Craven Street had been sufficiently extended allowing for the construction of ten new houses below the original houses: “ … four houses, Nos  21–24, were built on the west side, and six houses, Nos  25–30, on the east side of the way ”[11] In a note in the same report, the new numbering system is explained  “The houses in the street, which had previously been numbered consecutively down the west side and up the east side, were then renumbered on the same system to include the additional houses ”[12] Because the new houses (21-24) on the west side were built below the existing houses (1-20), houses 1-20 retained their original numbering 
Figure 4  Craven Street 1799  (Richard Horwood’s Map of London, Westminster and the Borough of Southwark 1799, Motco Enterprises Limited, motco com)
One would think that the numbers of the sixteen original houses on the east side, Nos  21 – 36, would simply increase by ten with the addition of the ten new houses, but such was not the case; they increased by nine  How could that be? The only possible explanation is that No  21 of the original houses was demolished to make way for the construction of the northernmost of the six new houses on the east side (No  30)  Evidence of No  21’s demolition appears in the lease granted to Charles Owen by William, 7th Baron Craven, in 1792, which describes No  22 as: “All that messuage in Craven Street late in the occupation of Francis Deschamps undertaker … being the Southernmost house in the Old Buildings on the East Side of the said Street numbered with the No  22 ”[13] The lease describes No  22 as being the southernmost house in the old buildings on the east side of Craven Street  Clearly the house previously at No  21 did not exist when the lease granted to Charles Owen was written in 1792 as it used to be the southernmost house  It is also worth noting that in 1790, The London Directory listed Jacob Life at No  21 (original numbering)  In 1791-2, it listed him at No  6  With No  21 vacated, it would allow for its demolition and the construction of the tenth new house  By utilizing lot No  21 for the new construction, only nine additional lots were needed to build the ten houses, hence, Margaret Stevenson’s former residence at 27 became 36 (27 + 9) in the renumbering and not 37 
For nearly a century and a half after Franklin departed London for America in March of 1775 the scales were tipped heavily in favor of his residence having been No  7 Craven Street  As early as 1807 in London; Being An Accurate History And Description Of The British Metropolis And Its Neighborhood, Volume 4, one would have read: “In Craven Street is a house, No  7, remarkable for having been the residence of Dr  Benjamin Franklin [14] In 1815, the identical phrase appeared in The Beauties of England and Wales [15] After 23 editions of not mentioning Franklin, his name finally appeared in the 24th edition of The Picture of London in 1826: “The house, No  7, Craven Street, in the Strand, was once the residence of Dr  Benjamin Franklin ”[16] In 1840, Jared Sparks referred to Franklin’s Craven Street residence appearing in London guide books in his voluminous The Works of Benjamin Franklin: “In the London Guide Books, ‘No  7, Craven Street,’ is still indicated as the house in which Dr  Franklin resided ”[17] In 1846, George Gulliver F R S , in his book, The Works of William Hewson, wrote: “She [Polly] had been upon terms of the warmest friendship with Dr  Franklin
Figure 5  No  7 Craven Street with Memorial Tablet  (Photo courtesy of British History Online, and the Survey of London)
since she was eighteen years of age  That eminent philosopher resided with her mother, Mrs  Margaret Stevenson, at No  7, Craven Street, Strand, during the fifteen years of his abode in London ”[18] Guide books mentioning Franklin at No  7 continued to proliferate throughout the century: Handbook for London; Past and Present, Volume I (1849);”[19] Handbook for Modern London (1851);”[20] The Town; Its Memorable Characters and Events (1859);”[21] London and Its Environs (1879) [22] There was an anomaly when London In 1880 Illustrated With Bird’s-Eye Views of the Principal Streets, Sixth Edition (1880) placed Franklin at 27 Craven street [23] The anomaly lasted for six years until his place of residence was changed to No  7 in the revised edition, London  Illustrated by Eighteen Bird’s-Eye Views of the Principal Streets (1886) [24] London Past and Present; Its History, Associations, and Traditions, Volume 1 (1891), copied the 1849 Handbook for London almost word-for-word and included, “The house is on the right from the Strand ”[25] In October of 1867, The Society of Arts in London declared that: “In order to show how rich the metropolis is in the memory of important personages and events, which it would be desirable to mark by means of tablets on houses, the Council have caused an alphabetical list to be prepared, … ”[26] Franklin had been elected a corresponding member to the Society in 1756 and was a popular choice among Council members deciding who they were to memorialize [27] By January of 1870, a tablet honoring him was affixed to the house they believed to have been his residence while in London, No  7 Craven Street in the Strand on the west side of the street [28] A majority of historians writing about Franklin in the nineteenth and early twentieth century placed him at No  7: O  L  Holley, The Life of Benjamin Franklin (1848); E  M  Tomkinson, Benjamin Franklin (1885); John Torrey Morse, Benjamin Franklin (1891); Paul Elmer More, Benjamin Franklin (1900); John S  C  Abbot, Benjamin Franklin (1903); Sydney George Fisher, The True Benjamin Franklin (1903)  A notable exception is D  H  Montgomery’s His Life Written by Himself published in 1896  He has Franklin at No  27 Craven Street  It seems then that depending upon the source, Franklin was thought to have lived at either No  7 or No  27, but not both, the overwhelming majority favoring No  7  As late as 2011, Franklin is still mentioned as living at No  7 [29]
In 1913, No  7 was scheduled to be torn down  An article in the March 1914 edition of The Book News Monthly, describes the situation:
As is well known to informed American pilgrims, it has been possible for all admirers of the famous philosopher and statesman to pay their respects to his memory before that house, No  7 Craven Street, just off the Strand, which was his chief home during his two sojourns in the British capital, but even as these lines are being written the London newspapers are recording that that interesting shrine is soon to be pulled down to make room for a restaurant  It is some mitigation of this misfortune to remember that at the most the Craven Street house was nothing more than a reproduction of the one in which Franklin had his suite of four rooms, for the structure has been rebuilt since Franklin’s time  When, then, some one makes a piteous plea that at least the philosopher’s bedroom shall be preserved, the soothing answer is that the apartment in question is only a replica of that in which the illustrious American enjoyed his well-earned slumbers in 1757-62 and 1764-75  The restaurant-builder, however, with an eye doubtless to possible American patronage, has assured the world that every effort will be made to preserve as much as possible of the entire structure [30]
Concerned with the possible demolition of Franklin’s residence, the Royal Society of Arts (formerly the Society of Arts[31]) initiated an inquiry into the matter [32] The London County Council, having taken over the responsibility of placing memorial tablets on notable houses from the Royal Society, was charged with the investigation  It ultimately fell to Sir George Laurence Gomme, a clerk to the Council, to come up with a response  A few years earlier Sir George had discovered Margaret Stevenson residing at No  27 Craven Street in the Westminster Rate Books  He must have wondered why No  7 on the west side of Craven Street was being celebrated as Franklin’s residence when the evidence clearly showed otherwise 
Sir George and his staff examined the various London directories discussed earlier and came up with a novel explanation for the discrepancy  They concluded that there had been two numbering systems on Craven Street  An anonymous author echoes Sir George’s conclusion about the two numbering systems in an article in The Journal of the Royal Society of Arts:
…an inspection of the directories of that time proves that there were at least two systems of numbering in Craven Street before the erection of the additional houses  According to one of these the numbers started from the top (Strand end) on the west side of the street, and ran down to the bottom to No  20, then crossed over and went back to the Strand along the east side – 21 to 36  According to the other system, the east side of the street was numbered from the bottom upwards, starting at No 1  This was not apparently in general use, but there is evidence that this numbering was at all events occasionally used 
The evidence of these two systems of numbering, and for believing that Mrs  Stevenson’s house was first No  7 under the oldest system, next No  27 under the second system, and finally No  36 under the latest and existing system, is to be found in the various directories and the Westminster rate-books [33]
The “evidence” mentioned above consisted of The London Directory’s listing of Brown & Whiteford at No  9: “The rate-books for 1781 and 1786 show the house next but one to the north of Mrs  Stevenson’s house as in the occupation of Brown and ‘Whiteford,’ while the old directories mention the business of the firm as wine merchants, and give their address as 9, Craven Street – then a little later, down to 1791, as 29, Craven Street  Curiously enough, in the years 1778 to 1780, or 1781, Lowndes gives it as No  9, and Kent as 29 ”[34] Ignoring Kent’s Directory having Brown and Whiteford as 29 and The London Directory (Lowndes) having Brown and Whiteford “a little later” as 29, and knowing that Mrs  Stevenson lived two doors south of them, Sir George concluded that her house must have been numbered 7, even though there is no listing in any of the directories of her residence ever being No  7  He surmised that the No  7 on the west side of Craven Street with the memorial tablet thought to have been Franklin’s residence had simply been confused with number 7 (27) on the east side  Again from The Journal of the Royal Society of Arts:
Taking all the evidence together, there cannot be any doubt whatever that Mrs  Stevenson’s house, in which Franklin lodged, was the house two doors north from Craven Court, first numbered 7, afterwards 27, and finally 36, and consequently that the house in which Franklin lived was that now numbered 36, not the one now numbered 7, on which the tablet is placed [35]
A response to The Royal Society of Arts was issued: “… the London County Council … informed the Society that it had made a mistake and that No  36 Craven street was the building that deserved commemoration ”[36] The Society accepted the Council’s conclusion, and despite assurances of preservation by the restaurant builder, No  7 was torn down the following year 
Sir George’s assertion “that Mrs  Stevenson’s house, in which Franklin lodged, was the house two doors north from Craven Court” was correct, however, his assertion that it was “first numbered 7, afterwards 27”, was not  It was only by association with the errant entry of Brown & Whiteford at No  9 from 1776-1782 in The London Directory that Mrs  Stevenson’s address was conjured to be No  7  The problem with associating her address exclusively with that of Brown & Whiteford at No  9 during those years is that, as previously demonstrated, The London Directory also listed four other Craven Street residents, Bond, Rowles, Sneyd, and Michie, who’s addresses did conform to the numbering system in The Westminster Rate Books  If Brown & Whiteford at No  9 was indicative of a numbering system different from The Westminster Rate Books, Bond, Rowles, Sneyd, and Michie would have been listed as Nos  10, 11, 12, and 15, respectively  So on one hand Sir George was relying on the Westminster Rate Books to establish Mrs  Stevenson at No  27 and on the other hand he was dismissing the Westminster Rate Books to establish her at No  7  Instead of using the anomalous listing of Brown & Whiteford at No  9, he could have just as easily, and more logically, used the Bond et al  listings, or the post-1782 Brown & Whiteford listing in the London Directory at No  29 to establish Mrs  Stevenson at No  27  Even if there had been two numbering systems, his assertion that No  27 was first numbered 7 would still be false  The earliest numbering system was the Westminster Rate Books dating from the early 1730s when the houses were constructed  Brown & Whiteford at No  9 didn’t appear until 46 years later and then only for a brief period 
There is ample evidence in Franklin’s correspondence and in a memoir by Polly Hewson (Mrs  Stevenson’s daughter) that Benjamin and Mrs  Stevenson lived in not one, but two houses on Craven Street  On July 6, 1772, Polly wrote to Benjamin from her house at Broad Street North in London: “My Mother I must tell you went off last friday week, took our little Boy with her and left Mr  Hewson [Polly’s husband, William] the care of her House [27 Craven Street]  The first thing he did was pulling down a part of it in order to turn it to his own purpose, and advantage we hope  This Demolition cannot affect you, who at present are not even a Lodger [Benjamin was traveling at the time], your litterary apartment remains untouch’d, the Door is lock’d …”[37] In a memoir about her husband written after his death Polly writes: “He [William Hewson] began his Lectures Sept  30, 1772, in Craven-street, where he had built a Theatre adjoining a house which he intended for the future residence of his family ”[38] On October 7, 1772, Benjamin wrote to his son William: “I am very well  But we [Mrs  Stevenson and I] are moving to another House in the same street; and I go down tomorrow to Lord LeDespencer’s to [stay a] Week till things are settled ”[39] To his son-in-law, Richard Bache, on the same day he wrote: “We are moving to another House in the [street] leaving this to Mr  Hewson ”[40] Writing to a friend on October 30, 1772 he explained: “I should sooner have answered your Questions but that in the Confusion of my Papers, occasioned by removing to another House, I could not readily find the Memorandums …”[41] On November 4, 1772 Benjamin informed his wife Deborah of the move  “We are removed to a more convenient House in the same street, Mrs  Stevenson having accommodated her Son-in-Law with that we lived in  The Removing has been a troublesome Affair, but is now over ”[42]
An agreement had been struck between the parties  Margaret and Benjamin would move to another house on Craven Street and allow Polly and William to move into No  27, the large yard behind the house being spacious enough to accommodate the anatomy school William wished to build [43] Perhaps the idea was inspired by Margaret’s next-door neighbor at No  26, Dr  John Leake, a man-midwife and founder of the Westminster Lying-in Hospital, who had built a theater adjoining his residence in which he practiced anatomy and taught midwifery [44]
After Margaret and Benjamin vacated No  27, Polly, William, their son William Jr , and William’s younger sister, Dorothy Hewson, took up residence there [45] In the 1773 Westminster Rate Books for Craven Street, Mrs  Stevenson’s (Stephenson in the Rate Books) name has been crossed out and replaced with “William Hewson ”[46] Further proof that the Hewsons had indeed moved into 27 Craven Street has been confirmed by the discovery of human and animal remains buried in the basement of No  36 (formerly No  27 and now the Benjamin Franklin House), a by-product of the dissections that took place at William’s anatomy school [47]
So what house on Craven Street did Mrs  Stevenson and Benjamin move into after vacating No  27? An examination of the Westminster Rate Books for the years 1774 and 1775 reveal them living not at No  7 on the west side of Craven Street as one might expect from the overwhelming consensus of nineteenth century guidebooks and biographies, but surprisingly at No  1 [48] The controversy of No  7 being torn down was all for naught as it had never been Franklin’s residence  Sir George was correct on that point  Unfortunately, No  1 was torn down as well in the early part of the twentieth century  The first time No  1 is mentioned as Franklin’s second residence is in the Survey of London: Volume 18, St Martin-in-The-Fields II: the Strand published by the London County Council in 1937, ironically the same County Council that had declared No  36 as Franklin’s only residence twenty-four years earlier 
From 1748 until 1772 Margaret ‘Stephenson’ occupied this house [No  27 (36)], and it was there that Benjamin Franklin settled after his arrival in London in 1757 as Agent to the General Assembly of Pennsylvania … In October, 1772, Mrs  Stevenson and Franklin removed to No  1, Craven Street (now demolished), and No  36 was for the next two years occupied by William Hewson, surgeon, who had married Mary Stevenson [49]
In the spring of 1774, William Hewson died unexpectedly of septicemia two weeks after cutting himself while dissecting a cadaver  Polly was left to care for their two young sons and was pregnant with a daughter she would give birth to in August of the same year  Is it possible that Margaret and Benjamin moved back into No  27 to assist Polly after the death of her husband as suggested in The Americanization of Benjamin Franklin?[50]
If the Westminster Rate Books are to be believed, the answer is no  For the year 1774, the Rate Books list Margaret Stevenson at No  1 and William Hewson at No  27  For the year 1775, they list Margaret Stevenson at No  1 and Magnus Falkner (Falconer/Falconar) at No  27  Magnus was William’s assistant at the anatomy school and fiancé to William’s sister, Dorothy  On his death bed, William instructed Polly, “let Mr  Falconar be my successor ”[51] Magnus would immediately take over the running of the anatomy school and continue William’s unfinished research  Four months later, he and Dorothy would marry [52] Essentially only two things changed at 27 Craven Street after William’s death: Polly gave birth to her daughter, and Magnus replaced William as the lease holder, so even if Margaret and Benjamin had wished to move back into No  27, there would have been no room for them  It is also interesting to note that considering the multiple times Benjamin wrote of his move out of No  27 (and complained of it), he never once mentioned moving back into No  27 in any of his correspondence after Mr  Hewson’s death 
Figure 6  No  36 Craven Street  (Photo courtesy of David Ross, britainexpress com)
In sum, based on the Westminster Rate Books[53] and Franklin’s correspondence, Mrs  Stevenson is known to have resided at No  27 (36) Craven Street from 1748 to 1772  It follows that, aside from the two years Franklin spent in Philadelphia from 1762 to 1764, he resided there from 1757 to 1772  Franklin’s correspondence also reveals that in the autumn of 1772, he and Mrs  Stevenson moved to another house on Craven Street  The 1773 Westminster Rate Books show her name crossed off at No  27 and William Hewson’s inserted  The following year the Rate Books list her at No  1 Craven Street  Evidence for Mrs  Stevenson and Benjamin remaining at No  1 after William’s death appears in the Westminster Rate Books for 1775 which have Mrs  Stevenson still residing at No  1 and Magnus Falkner residing at No  27  Further evidence can be construed from the lack of any mention of a move back into No  27 in Franklin’s correspondence  Despite the many theories one could devise as to why Franklin was thought to have lived at No  7 Craven Street by so many guide books and Franklin biographers of the nineteenth century, one thing is certain; at some point after Franklin’s departure to America in March of 1775, and no later than 1807, someone mistakenly associated him with No  7 on the west side of Craven Street, and it soon became his de facto residence  Credit must go to D  H  Montgomery in 1896 and Sir George in 1913 for setting the record partially straight by placing Franklin at No  27(36)  In 1937, the London County Council gave us the first accurate account of Franklin’s residences on Craven Street in the Survey of London at No  27(36) and No  1  It has been shown conclusively that No  27 was never previously numbered 7  It was, however, renumbered 36 in 1792 after ten additional houses were built at the southern end of the street and remains No  36 to this day 
[1] “Craven Street and Hungerford Lane”, in Survey of London: Volume 18, St Martin-in-the-Fields II: the Strand, ed  G H Gater and E P Wheeler (London, 1937), 27-39, Early History of the Site 
http://www british-history ac uk/survey-london/vol18/pt2/pp27-39
[2] “England, Westminster Rate Books, 1634-1900,” from database with images, Craven Street – 1735, FamilySearch from database by FindMyPast and images digitized by FamilySearch; citing Westminster City Archives, London 
[3] Ibid , Craven Street – 1748 
[4] The Statutes at Large, From Magna Charta to the End of the Eleventh Parliament of Great Britain  Anno 1761 Continued, Vol  XXVII, ed  Danby Pickering, (Cambridge, John Archdeacon, 1767), 96 
[6] James Raven, Publishing Business in Eighteenth-Century England, (Woodbridge: The Boydell Press, 2014), 201 
[7] The London Directory For the Year 1776, Ninth Edition, (London: T  Lowndes, 1776), title page 
[8] Kent’s Directory For the Year 1778, Forty-Sixth Edition, (London: Richard and Henry Causton, 1778), title page 
[9] A listing in Kent’s Directory for the Year 1882 on p  28 reveals, “Brown Sarah, Leather-seller, 1, Westmoreland-buildings, Aldersgate-street”, and in Kent’s Directory for the Year 1883 on p  175, “Whiteland Mary, Wine & Brandy Mercht  Jermyn-str  St  James ”
[10] “The Papers of Benjamin Franklin,” Sponsored by The American Philosophical Society and Yale University, Digital Edition by The Packard Humanities Institute, 22:263a 
http://franklinpapers org/franklin
Mrs  Stevenson wrote to Benjamin Franklin a letter from her new home at 75 Northumberland Court on November 16, 1775: “In this Court I have a kind friend, Mr  Lechmoen he comes and seats with me and talks of you with a hiy regard and friendship ”
[11] Survey of London, Early History of the Site 
[12] Survey of London, Footnotes/n 10 
[13] Survey of London, Historical Notes/No  31 
[14] David Hughson, LL D , London; Being An Accurate History And Description Of The British Metropolis And Its Neighbourhood, To Thirty Miles Extent, From An Actual Perambulation, Vol  IV, (London: W  Stratford, 1807), 227 
[15] The Reverend Joseph Nightingale, The Beauties of England and Wales: Or, Original Delineations, Topographical, Historical, and Descriptive, of Each County, Vol  X, Part III, Vol  II (London: J  Harris; Longman and Co ; J  Walker; R  Baldwin; Sherwood and Co ; J  and J  Cundee; B  and R  Crosby and Co ; J Cuthell; J  and J  Richardson; Cadell and Davies; C  and J  Rivington; and G  Cowie and Co , 1815), 245 
[16] John Britton, F S A  & Co , ed , The Original Picture of London, Enlarged and Improved: Being A Correct Guide For The Stranger, As Well As For the Inhabitant, To The Metropolis Of The British Empire Together With A Description Of The Environs, The Twenty-Fourth Edition (London: Longman, Rees, Orme, Brown, and Green, 1826), 479 
[17] Jared Sparks, The Works of Benjamin Franklin, Vol  VII, (Philadelphia: Childs & Peterson, 1840), 151 
[18] George Gulliver, F R S , The Works of William Hewson, F  R  S , (London: Printed for the Sydenham Society, MDCCCXLVI), xx 
[19] Peter Cunningham, Handbook for London; Past and Present, Vol  I, (London: John Murray, 1849), 245 
[20] F  Saunders, Memories of the Great Metropolis: or, London, from the Tower to the Crystal Palace, (New York: G P  Putnam, MDCCCLII), 138 
[21] Leigh Hunt, The Town; Its Memorable Characters and Events, (London: Smith, Elder and Co , 1859), 185 
[22] K  Baedeker, London and Its Environs, Including Excursions To Brighton, The Isle of Wight, Etc : Handbook For Travelers, Second Edition, (London: Dulau and Co , 1879), 133 
[23] Herbert Fry, London In 1880 Illustrated With Bird’s-Eye Views of the Principal Streets, Sixth Edition, (New York: Scribner, Welford, & Co , 1880), 50 
[24] Herbert Fry, London  Illustrated By Eighteen Bird’s-Eye Views of the Principal Streets, (London: W  H  Allen and Co , 1886), 40 
[25] Henry B  Wheatley, F S A , London Past and Present; Its History, Associations, and Traditions, Vol  1, (London: John Murray, New York: Scribner & Welford, 1891), 473 
[26] The Journal of the Society of Arts, Vol  XV, No  778, (October 18, 1867): 717 
[27] D  G  C  Allen, “Dear and Serviceable to Each Other: Benjamin Franklin and the Royal Society of Arts,” American Philosophical Society, Vol  144, No  3, (September 2000): 248-249 
Franklin was a corresponding member in 1756 because he was still residing in Philadelphia  He became an active member the following year when he moved to London 
[28] The Journal of the Society of Arts, Vol  XVIII, No  894, (Jan  7, 1870): 137 
“Since the last announcement, the following tablets have been affixed on houses formerly occupied by – Benjamin Franklin, 7 Craven-street, Strand, W C ”
[29] Franklin in His Own Time, eds  Kevin J  Haytes and Isabelle Bour, (Iowa City, University of Iowa Press, 2011), xxxvii 
“Takes lodgings with Margaret Stevenson at No  7 Craven Street ” It is unknown if the editors are referring to No  7 on the west side of Craven Street or No  36 on the east side using Sir George’s explanation of No  36 being previously numbered 7 
[30] Henry C  Shelly, “American Shrines on English Soil, III  In the Footprints of Benjamin Franklin,” in The Book News Monthly, September, 1913 to August, 1914, (Philadelphia: John Wanamaker, 1914), 325 
[31] The Journal of the Royal Society of Arts, Vol  LVI, No  2,880, (Jan  31, 1908): 245 
http://babel hathitrust org/cgi/pt?id=mdp 39015058423073;view=1up;seq=251
“His Majesty the King, who is Patron of the Society, has granted permission to the Society to prefix to its title the term ‘Royal,’ and the Society will consequently be known in future as the ‘Royal Society of Arts ’”
[32] Nineteenth Annual Report, 1914, of the American Scenic and Historic Preservation Society, (Albany: J  B  Lyon Company, 1914), 293 
http://babel hathitrust org/cgi/pt?id=wu 89072985302;view=1up;seq=4;size=150
[33] The Journal of the Society of Arts, Vol  LXII, No  3,183, (Nov  21, 1913): 18 
http://babel hathitrust org/cgi/pt?id=mdp 39015058422968;view=1up;seq=26
[36] Allen, “Dear and Serviceable,” 263-264 
[37] Papers of Benjamin Franklin, 19:20 
[38] Thomas Joseph Pettigrew, F  L  S , Memoirs of the Life and Writings of the Late John Coakley Lettsom With a Selection From His Correspondence, Vol  I, (London: Nichols, Son, and Bentley, 1817), 144 of Correspondence 
[39] Papers of Benjamin Franklin, 19:321b 
[40] Ibid , 19:314 
[41] Ibid , 19:353a 
[43] Simon David John Chaplin, John Hunter and the ‘museum oeconomy’, 1750-1800, Department of History, King’s College London  Thesis submitted for the degree of Doctor of Philosophy of the University of London , 202 
“Following Falconar’s death [1778] the lease [27 Craven Street] was advertised, and the buildings were described as:
A genteel and commodious house, in good Repair, with Coach-house and Stabling for two Horses…consisting of two rooms and light closets on each floor, with outbuildings in the Yard, a Museum, a Compleat Theatre, and other conveniences  (Daily Advertiser, 27 August 1778)”
[44] Simon Chaplin, “Dissection and Display in Eighteenth-Century London,” in Anatomical Dissection in Enlightenment England and Beyond: Autopsy, Pathology and Display, ed  Dr  Piers Mitchell, (Burlington: Ashgate Publishing Company, 2012), 108 
“Given that a nearby building at 35 [ No  26 in Franklin’s time] was occupied by the man-midwife John Leake, who advertised lectures – including lessons in the art of making preparations – at his ‘theatre’ between 1764 and 1788, it is possible that some facilities were shared  In both cases, however, the buildings [Leake’s residence at No  26 and Hewson’s residence next door at 27] served a dual function as domestic accommodation and as sites for lecturing and dissection ”
[45] George Gulliver, F R S , The Works of William Hewson, F  R  S , (London: Printed for the Sydenham Society, MDCCCXLVI), xviii 
[46] Westminster Rate Books, Craven Street – 1773, courtesy of the City of Westminster Archives 
[47] S W  Hillson et al , “Benjamin Franklin, William Hewson, and the Craven Street Bones,” Archaeology International, Vol  2, (Nov  22, 1998): 14-16 
http://dx doi org/10 5334/ai 0206
[48] Westminster Rate Books, Craven Street – 1774, 1775, courtesy of the City of Westminster Archives 
[49] Survey of London, Historical Notes/No  36, Craven Street (not sourced) 
[50] Gordon S  Wood, The Americanization of Benjamin Franklin, (New York: The Penguin Press, 2004), 261 
[51] Pettigrew, Memoirs, 146 of Correspondence 
[52] http://founders archives gov/documents/Franklin/01-22-02-0178, note 7  “Falconar married Hewson’s sister five months after the Doctor’s death; most of the Craven Street circle attended the wedding, and BF gave away the bride: Polly to Barbara Hewson, Oct  4, 1774, APS” (American Philosophical Society); “England Marriages, 1538–1973 ,” database, FamilySearch (https://familysearch org/ark:/61903/1:1:V52W-TGS : accessed September 15, 2015), Magnus Falconar and Dorothy Hewson, September 12, 1774; citing Saint Martin In The Fields, Westminster, London, England, reference ; FHL microfilm 561156, 561157, 561158, 942 B4HA V  25, 942 B4HA V  66 
[53] I chose to rely on the Westminster Rate Books for the numbering system on Craven Street  The books were consistent throughout the eighteenth century in the ordering of residents on the street and were used as the basis for the 1792 re-numbering  For the most part, commercial directories aligned with them as well  If by chance a directory didn’t initially align, it would inevitably produce future editions that did 
Benjamin Franklin, Benjamin Franklin House, London
More from David Turnquist
If one looked into Benjamin Franklin’s time on Craven Street, they might   
I think it’s very ironic that on the street maps included in your excellent article, Craven Street is so close to Scotland Yard  Because following the back and forth juxtapositions of numbers 7, 27 and 36 Craven Street (throw in 75 Northumberland Court and 1 Craven Street, too) was a case that could confound Sherlock Holmes 
Excellent job of deciphering street renumbering material spanning sixty years, including that of a wrong house number (# 7) being erroneously identified and then perpetuated in subsequent street map printings  It’s gratifying at least to know that the present day #36 Craven Street is the correct house for Ben Franklin tourists to visit  Except for #1 Craven Street for the last three years Franklin was in London, but we won’t get into that 
Again, excellent article, David!
Context 2: '无锡速芯微电子有限公司是一家集芯片 研发，销售和服务于一体的国家高新技 术企业，为客户提供高性能，高集成 度，极致体验的全协议快充芯片。 无锡速芯微电子有限公司 FastSOC Microelectronics Co ,Ltd  销售联系方式： 联系人：顾先生 手机：1800 185 3071 邮箱：gpp@fastsoc com 网址：www fastsoc com 地址：无锡市新吴区菱湖大道200号中国物联网国际创新园E-503室 顾工微信号 速芯微公众号 免责声明：本文所述方法、方案均供客户参考，用于提示或者展示芯片应用的一种或者多种方式，不作为最终产品的实际方案。文中所描述的功能和性能指标在实 验室环境下测试得到，部分可以提供第三方测试报告，但是不保证客户产品上能获得相同的数据。本文信息只作为芯片使用的指导，不授权用户使用本公司或者其 他公司的知识产权。本文信息只作为芯片使用的指导，不承担因为客户自身应用不当而造成的任何损失。 **文中信息仅供参考，详情请联系我司获取最新资料” 无锡速芯微电子有限公司 FastSOC Microelectronics Co ,Ltd  产品手册 2023年 
新品快览 FS312A：PD3 0 诱骗- FS312A支持PD2 0/PD3 0最高诱骗电压：20V - FS312AE支持PD2 0/PD3 0 最高诱骗电压：20V支持Emarker模拟功能 - 封装：SOT23-5 VBUS CC1 CC2 DM DP 用电电路 4 7K 0 47uF R C C 1 V D D F U N C C C 2F S 3 1 2 B D M D P EP GND 应用图 FS8628：A+C快充协议CC2 CC1 VBUS CC2 CC1 FS312A FUNC GND VDD 4 7K GND R 用电电路 1uF GND 应用图 多口极简方案 FS8611SP*2+CCM-8611SP-A+7533B-T 双C智能降功率方案 FS8611S USB-C AC-DC 双变压器 7533B-T CCM-8611SP-A FS8611S USB-C 采用2颗FS8611SP搭配CCM-8611SP-A （MCU），7533B-T配合工作 - 支持多种协议 - 支持I2C控制 - 任意单 C 的为 35W - 双 插 降 功 率 ， 三 档 功 率 智 能 配 置：27 4W+7 4W；17 4W+17 4W； 27 4W - BOM极简，成本低 FS312B：PD3 1 诱骗FS8611K*2+CCM-8611K-A+7550B-T 双C方案 - FS312BL支持PD2 0/PD3 0/PD3 1/第三方协议最高诱骗电压：20V - FS312BLE支持PD2 0/PD3 0/PD3 1/第三方协议最高诱骗电压：20V支持Emarker模拟功能 - FS312BH支持PD2 0/PD3 0/PD3 1/第三方协议最高诱骗电压：48V - FS312BHE支持PD2 0/PD3 0/PD3 1/第三方协议最高诱骗电压：48V 支持Emarker模拟功能 - 封装：DFN2x2-6L - 兼容兼容BC1 2、Apple2 4A、 QC2 0 Class A、QC3 0 Class A/B、 FCP、SCP、AFC、低压直充等 - 兼容Type-C PD2 0、Type-C PD3 0、 Type-C PD3



ground_truth: 1766.

prompt_front: Now, answer the following question based on the text below, only give me the answer and do not output any other words.

Question: 对于PD3.0协议，FS312BH支持的最高诱骗电压是多少？

Read the following text and answer briefly.

Context 2: 0 PPS、QC4 0协议 - 支持两路DP/DM - 支持CV/CC（分段CC）功能 - 支持定制PDO - 支持A+C双口工作，电压自动回5V - 支持FB/OPTO反馈 - 封装：QFN3x3-20L VPWR FB PowerSystem 100K GND R1 GND 19 VIN 17 FB FUNC1 FUNC2 20 15 18 13 PLUGIND VFB FS8628 QFN3x3-20L AGATE 47K 7 5K 47K 7 5K 1 16 8 7 3 4 5 6 10 9 11 CGATE CVBUS CC2 CC1 CDP CDM AVBUS DM DP ISP ISN 12 应用图 2 V3P3 100Ω 1u EP GND GND CVBUS TYPE- C CC2 CC1 CDP CDM CGND TYPE-A AVBUS DM DP 10n 200 AGND 5mΩ GND FS8611K USB-C AC-DC DC-DC 7550B-T CCM-8611K-A FS8611K USB-C 采用2颗FS8611K搭配CCM-8611K-A （MCU）工作，7550B-T配合工作 - 支持PD2 0/PD3 0/QC2 0/AFC/FCP - 支持PDO定制 - 任意单 C 的为 35W(可定制) - 双插18W（可定制15W/20W） - BOM极简，成本低 FS212C+ACM-212C-A+7550B-T 双C方案 FS212C USB-C AC-DC DC-DC 7550B-T ACM-212C-A FS8623B-A+C方案 AC-DC DC-DC FS8623B USB-A USB-C USB-A 采 用 1 颗 F S 2 1 2 C 搭 配 ACM-212C-A 工 作，7550B-T配合工作 - 支持PD2 0/PD3 0 - 支持PDO定制 - 任意单 C 的为20W - 双插7 5W回5V - BOM极简，成本低 采用一颗FS8623B实现A+C方案 - 兼容兼容Apple2 4A/低压直充 QC2 0 Class A/QC3 0 Class A/B/ FCP/SCP等 - 兼 容Type -C PD2 0 / PD3 0 / PD3 0PPS/QC4 0协议 - 支持PDO定制 - 双插回5V 
多口方案选型 产品选型 受电端芯片选型 速芯微现有多种多口的方案选择：A+C，C+C，C+C+A，C+C+C，C+C+A+A等方案。对于 A+C的方案，可使用1颗芯片实现，也可用多颗芯片来实现。 速芯微现有多种受电端诱骗芯片，客户可根据应用需求进行选择。 受电端诱骗芯片应用领域 筋膜枪 无线充 线材 无人机 产品型号 PD2 0 PD3 0 PD3 1 第三方协议 诱骗电压(V) 控制方式 内置Emarker 定制 封装 FS312A √ √ 5/9/12/15/20 电阻阻值 可变电压策略 SOT23-5 FS312AE √ √ 5/9/12/15/20 电阻阻值 √ (公头专用) 可变电压策略 SOT23-5 FS312BL √ √ √ √ 5/9/12/15/20 电阻阻值 可变电压策略 DFN2x2-6 FS312BLE √ √ √ √ 5/9/12/15/20 电阻阻值 √ (公头专用) 可变电压策略 DFN2x2-6 FS312BH √ √ √ √ 5/20/28/36/48 电阻阻值 可变电压策略 DFN2x2-6 FS312BHE √ √ √ √ 5/20/28/36/48 电阻阻值 √ (公头专用) 可变电压策略 DFN2x2-6 FS312LC √ √ √ 5/9/12 电阻阻值 可变第三方 协议 SSOP10 FS312HC √ √ √ 5/9/12/15/20 电阻阻值 可变第三方 协议 SSOP10 FS2711Q √ √ √ 任意设置 I2C √ QFN3x3-16 FS2711P √ √ √ 任意设置 I2C √ QFN3x3-16 FS2711PA √ √ 全协议 任意设置 I2C √ SSOP10 FS2711SW √ √ 全协议 SSOP10 FS512 √ √ 全协议 任意设置 I2C √ SSOP10 方案 类型 产品型号 单C 单A 双插 A+C方案 FS8623 20W（PPS）（可定制） A口全协议18w 5V共享3A FS8623B 20W（PPS）(可定制) A口全协议18w 5V共享3A FS8628 20W（PPS）(可定制) A口全协议18w 5V共享3A FS8611RPC+FS116DB 65W（PPS）(可定制) A口全协议18w A口：5V/2 4A C口：45W FS8628RC+FS116DB 35W(可定制) A口全协议18w A口：5V（BC1 2，Apple 2 4） C口：20W 方案类型 产品型号 单C1 单C2 C1/C2 C+C方案 FS8611RPB*2 30W(可定制) 30W(可定制) C1/C2：5V/3A(或5V/2 4A) FS8611GH*2 35W（可定制） 35W（可定制） C1/C2:18W（可定制） FS8628P*2 35W（可定制） 35W（可定制） C1/C2：17 4W可定制） FS8611KL*2 20W（可定制） 20W（可定制） C1/C2：5V/1 5 A FS8611PC*2 35W 35W C1/C2：18W FS8611BH*2 65W（可定制） 65W（可定制） C1：45W（可定制）C2：20W（可定制） FS8628RPC+FS8611RB 45W（可定制）） 36W （可定制）） C1：30W（可定制）C2：5V/1 5A（可定制） 方案类型 产品型号 单C1 单C2 单A C1+C2 C1/C2+A C1+C2+A C+C+A FS8611S*2+FS116DB 65W（可定制） 65W（ 可定制）） A口全协议18w 智能分配功率 45W+18W C1/C2：智能分配功率 A：18W（或5V1 5A） FS8612C+FS8628P 100W（可定制） 35W （可定制）） 20W C1：65W C2：20W C1+A：65W+20W C2+A：7 5W+7 5W C1：65W C2：7 5W A：7 5W 其他 
Source-TYPE C协议芯片选型 Source-TYPE A协议芯片选型 速芯微现有多种TYPE-C的快充协议芯片，支持多种协议，支持客户定制，多样化，满 足客户对TYPE C的各种快充需求。 速芯微现有多种TYPE A快充协议芯片，支持全协议，支持定制，满足客户对A口协议的各种需 求。速芯微的TYPE-A快充协议芯片的协议丰富，FS112系列拥有多种的型号；FS116D 系列带插入指示，可搭配TYPE-C快充协议芯片，实现A+C，A+C+C，A+A+C+C等多口方 案，协议丰富，其中FS116A一般用于插入指示使用 Source-TYPE A协议芯片引脚封装图 D+ VSS FB 1 2 3 FS112 6 5 4 D- VDD FUNC GATE VIN FUNC FB LED/PLUG_IN 1 2 3 4 5 FS116D 10 DM 9 8 7 6 DP CSP CSN VSS速芯微的各TYPE-C快充协议芯片之间可搭配使用，实现多口方案，更多详情请咨 询我司工作人员。 多口降功率专用快充协议芯片：FS8611RB，FS8611RC，FS8611RPB，FS8611RPC， FS8612CP。 带I2C快充协议芯片：FS8611S，FS8611SP 产品型号 BC1 2 Apple 2 4 QC2 0 QC3 0 AFC FCP SCP HISCP 大电流直充 封装 FS112 √ √ √ √ √ √ √ SOT23-6 FS112H √ √ √ √ √ √ √ √ √ SOT23-6 FS113 √ v √ √ √ √ √ √ √ SOT23-6 FS116DP √ √ √ √ √ √ √ √ SSOP10 FS116DB √ √ √ √ √ √ √ √ SSOP10 FS116E √ √ √ √ √ √ √ √ √ SSOP10 FS116A √ √ SSOP10 其他 可定制 PD2 0 PD3 0 PD3 0 PPS 第三方协议 反馈方式 MOS CV/CC 定制 封装 FS212C √ √ FB √ SOT23-6 FS212CM √ √ FB PMOS(可省) √ SOT23-6 FS212D √ √ √ FB √ SOT23-6 FS212DH √ √ √ FB √ SOT23-6 FS212DP √ √ √ FB PMOS √ SOT23-6 FS212DG √ √ √ FB PMOS √ SOT23-6 FS8611G √ √ FB PMOS(可省) √ SOP-8 FS8611K √ √ QC2 0/AFC/FCP FB PMOS(可省) √ SOP8 FS8611J √ √ √ 全协议 FB PMOS(可省) √ SOP8 FS8611B √ √ √ 全协议 FB PMOS(可省) √ SSOP10 FS8611RB √ √ 全协议 FB PMOS √ SSOP10 FS8611RC √ √ 全协议 FB PMOS √ SSOP10 FS8611S √ √ √ 全协议 FB PMOS √ SSOP10 FS8611PP √ √ √ 全协议 FB PMOS √ SSOP10 FS8611BP √ √ √ 全协议 FB PMOS(可省) √ SSOP10 FS8611RPB √ √ √ 全协议 FB PMOS √ SSOP10 FS8611RPC √ √ √ 全协议 FB PMOS √ SSOP10 FS8611SP √ √ √ 全协议 FB PMOS(可省) SSOP10 FS8612 √ √ √ 全协议 OPTO PMOS √ √ SSOP16 FS8612B √ √ √ 全协议 FB PMOS √ √ SSOP16 FS8612BP √ √ √ 全协议 FB PMOS √ √ SSOP16 FS8612C √ √ √ 全协议 FB/OPTO PMOS √ √ QFN4x4-16 FS8612CP √ √ √ 全协议 FB/OPTO PMOS √ √ QFN4x4-16 
'
Context 3: Current address: Division of Brain Sciences, Department of Medicine, Imperial College London, London, United Kingdom 
In a variety of species, reduced food intake, and in particular protein or amino acid (AA) restriction, extends lifespan and healthspan  However, the underlying epigenetic and/or transcriptional mechanisms are largely unknown, and dissection of specific pathways in cultured cells may contribute to filling this gap  We have previously shown that, in mammalian cells, deprivation of essential AAs (methionine/cysteine or tyrosine) leads to the transcriptional reactivation of integrated silenced transgenes, including plasmid and retroviral vectors and latent HIV-1 provirus, by a process involving epigenetic chromatic remodeling and histone acetylation  Here we show that the deprivation of methionine/cysteine also leads to the transcriptional upregulation of endogenous retroviruses, suggesting that essential AA starvation affects the expression not only of exogenous non-native DNA sequences, but also of endogenous anciently-integrated and silenced parasitic elements of the genome  Moreover, we show that the transgene reactivation response is highly conserved in different mammalian cell types, and it is reproducible with deprivation of most essential AAs  The General Control Non-derepressible 2 (GCN2) kinase and the downstream integrated stress response represent the best candidates mediating this process; however, by pharmacological approaches, RNA interference and genomic editing, we demonstrate that they are not implicated  Instead, the response requires MEK/ERK and/or JNK activity and is reproduced by ribosomal inhibitors, suggesting that it is triggered by a novel nutrient-sensing and signaling pathway, initiated by translational block at the ribosome, and independent of mTOR and GCN2  Overall, these findings point to a general transcriptional response to essential AA deprivation, which affects the expression of non-native genomic sequences, with relevant implications for the epigenetic/transcriptional effects of AA restriction in health and disease 
Copyright: © 2018 De Vito et al  This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited 
Data Availability: All relevant data are within the paper and its Supporting Information files  RNAseq data are available in the ArrayExpress database under the accession number E-MTAB-6452 
Funding: This study was funded by the Ajinomoto Innovation Alliance Program, (AIAP; https://www ajinomoto com/en/rd/AIAP/index html#aiap) (to M V S and D G), which is a joint research initiative of Ajinomoto Co , Inc , Japan  One of the authors [M B ] is an employee of Ajinomoto Co , and his specific roles are articulated in the ‘author contributions’ section  The commercial funder provided support in the form of salary for author [M B ] and some of the necessary research materials (medium for cell culture), but did not have any additional role in the study design, data collection and analysis, or preparation of the manuscript, and the authors had unrestricted access to the data  Due to a confidentiality agreement, the commercial funder participated only in the decision to publish the data obtained during the study, without any restriction 
Competing interests: This study was funded by Ajinomoto Co , Inc , Japan and one of the authors [M B ] is an employee of this commercial funder  No other employment or consultancy relationships exist with the commercial funder, and no patents, products in development, or marketed products result from this study  The authors declare that no competing interests exist and that the commercial affiliation of one of the authors does not alter the adherence of authors to all PLOS ONE policies on sharing data and materials 
In animals, excessive, insufficient, or imbalanced nutrient availability is known to strongly impact on phenotype and health, both short and long-term, and across generations [1, 2]  In particular, studies in yeast, animal models and humans have shown that reduced food intake, reducing either overall calories, or only sugars, proteins, or even single amino acids (AA), such as Methionine (Met), may extend lifespan and healthspan, and reduce the risk of cancer and other age-related diseases [3–9]  In addition, fasting or specific AA deprivation have shown potential therapeutic applications, owing to their ability to directly reduce the growth of some tumor types [10, 11], sensitize cancer cells to chemo- or immunotherapy [12, 13], and allow efficient hematopoietic stem cell engraftment   However, little is known about the specific processes and molecular mechanisms mediating the roles of nutrient restriction in human health and longevity 
A properly balanced diet in metazoans contains optimal amounts of a subset of AA, which cannot be synthetized de novo and are therefore named essential amino acids (EAAs)  In humans these include Met, Histidine (His), Isoleucine (Ile), Leucine (Leu), Lysine (Lys), Phenylalanine (Phe), Threonine (Thr), Tryptophan (Trp), and Valine (Val), while a few others are considered as semi-essential, such as Glutamine (Gln) and Tyrosine (Tyr) [15, 16]  Consistently, EAA deprivation triggers a cell-autonomous adaptive response, characterized by extensive metabolic and gene expression modifications, implementing biosynthetic, catabolic, and plasma membrane transport processes, aimed at reconstituting the full AA complement [17, 18]  The best known and conserved pathways responding to AA deprivation are triggered by mechanistic Target of Rapamycin Complex 1 (mTORC1) and General amino acid Control Non-derepressible 2 (GCN2) protein kinases [15, 19, 20]  Activation of mTORC1 requires in particular the presence of Gln, Arg and Leu, but also Met , which activate the kinase through sensors mainly acting upstream of Rag GTPases at lysosomal membranes   In turn, mTORC1 promotes cell growth, proliferation and anabolism upon activation, and translational attenuation and autophagy upon inhibition [19, 20] 
By contrast, GCN2 is activated by deprivation of any individual EAA, by means of its histidyl-tRNA synthetase-related domain, which binds uncharged tRNAs accumulating during AA limitation [23, 24]  Upon activation, GCN2 phosphorylates and inhibits its only known downstream target, namely the eukaryotic Initiation Factor 2 α (eIF2α), thereby initiating the Integrated Stress Response (ISR)  This leads to attenuation of general translation, and induction of a transcriptional/translational program, aimed at increasing stress resistance and restoring cell homeostasis, by upregulating a specific subset of genes, including Activating Transcription Factor 4 (ATF4) and C/EBP-Homologous Protein (CHOP) [25–27]  Thus, inhibition of mTORC1 and activation of GCN2 by AA restriction cooperate to attenuate general translation at the initiation step, increase catabolism and turnover, and enhance stress resistance to promote adaptation   However, how these processes eventually induce protective mechanisms against the alterations associated with aging, which include pervasive epigenetic and transcriptional changes [28, 29], remains largely unknown 
We previously reported the unexpected observation that prolonged deprivation of either Tyr, or of both Methionine and Cysteine (Met/Cys), triggers the selective and reversible reactivation of exogenous transcriptional units, including plasmids, retroviral vectors and proviruses, integrated into the genome and transcriptionally repressed by defensive mechanisms against non-native DNA sequences [30, 31]  This phenomenon was observed both in HeLa epithelial and ACH-2 lymphocytic human cells, and was independent of the transgene or provirus (Ocular Albinism type 1, OA1; Green Fluorescent Protein, GFP; Lysosomal-Associated Membrane Protein 1, LAMP1; Human Immunodeficiency Virus-1, HIV-1), or of the exogenous promoter driving their transcription, either viral (cytomegalovirus, CMV; Long Terminal Repeat, LTR) or human (Phospho-Glycerate Kinase 1, PGK1; Elongation Factor-1α, EF-1α)   Furthermore, this transgene reactivation response was not reproduced by serum starvation, activation of p38, or pharmacological inhibitors of mTOR (PP242 or rapamycin), sirtuins and DNA methylation  By contrast, it was induced by pan histone deacetylase (HDAC) inhibitors, and by selective inhibitors of class II HDACs   Consistently, we found that the mechanism responsible involves epigenetic modifications at the transgene promoter, including reduced nucleosome occupancy and increased histone acetylation, and is mediated in part by reduced expression of a class II HDAC, namely HDAC4  
These findings indicate that AA deprivation induces a specific epigenetic and transcriptional response, affecting the expression of newly-integrated exogenous transgenes and proviruses, and suggesting that endogenous sequences sharing similar structural and functional features may represent a transcriptional target as well [30, 31]  In particular, transposable elements, such as LTR-retrotransposons (or endogenous retroviruses, ERVs), are genomic “parasites” anciently-integrated into the genome, and silenced by epigenetic mechanisms of mammalian cells against the spreading of mobile elements, eventually becoming "endogenized" during evolution [32, 33]  This raises the question of whether their expression is also sensitive to AA restriction  In addition, it remains unclear whether or not the transgene reactivation response is related to specific AA deprivations, and most importantly which is the AA sensing/signaling pathway involved, in particular whether the GCN2 kinase is implicated  Thus, here we used the reactivation of silenced transgenes in cultured cells, as a model to investigate a novel molecular pathway induced by imbalanced EAA starvation, implicated in the epigenetic/transcriptional regulation of exogenous non-native DNA sequences and possibly of other endogenous anciently-integrated genomic elements 
HeLa human epithelial carcinoma, HepG2 human hepatocellular carcinoma and C2C12 mouse skeletal muscle cells were maintained in DMEM containing glutaMAX (Invitrogen) and supplemented with 10% FBS (Sigma), 100 U/ml penicillin G (Invitrogen), 100 mg/ml streptomycin (Invitrogen), at 37°C in a 5% CO2 humidified atmosphere  Cell lines carrying integrated and partially silenced transgenes were also maintained in 600–1000 μg/ml G418 
The C2C12 cell line was provided by ATCC  HeLa and HepG2 cells were obtained by Drs  F  Blasi and G  Tonon at San Raffaele Scientific Institute, Milan, Italy, respectively, and were authenticated by Short Tandem Repeat (STR) profiling, using the Cell ID System kit (Promega), according to the manufacturer’s instructions  Briefly, STR-based multiplex PCR was carried out in a final volume of 25 μL/reaction, including 5 μL Cell ID Enzyme Mix 5X, 2 5 μL Cell ID Primer Mix 10X and 3 ng of template DNA  The thermal cycling conditions were: 1 cycle at 96°C for 2 min, followed by 32 cycles at 94°C for 30 sec, 62°C for 90 sec, and 72°C for 90 sec, and 1 cycle at 60°C for 45 sec  The following STR loci were amplified: AMEL, CSF1PO, D13S317, D16S539, D21S11, D5S818, D7S820, TH01, TPOX, vWA  Fragment length analysis of STR-PCR products was performed by Eurofins Genomics, using standard procedures of capillary electrophoresis on the Applied Biosystems 3130 XL sequencing machine, and assessment of the STR profile was performed at the online STR matching analysis service provided at http://www dsmz de/fp/cgi-bin/str html 
Stable cell clones, expressing myc-tagged human OA1 (GPR143) or GFP transcripts, were generated using pcDNA3 1/OA1myc-His or pcDNA3 1/EGFP vectors   Briefly, HeLa, HepG2 and C2C12 cells were transfected using FuGENE 6 (Roche) and selected with 800, 1000, and 650 μg/ml of G418 (Sigma), respectively, which was maintained thereafter to avoid loss of plasmid integration  G418-resistant clones were isolated and analyzed for protein expression by epifluorescence and/or immunoblotting 
Full DMEM-based medium, carrying the entire AA complement, and media deprived of Met/Cys (both AAs), Met (only), Cys (only), Alanine (Ala), Thr, Gln, Val, Leu, Tyr, Trp, Lys and His were prepared using the Nutrition free DMEM (cat #09077–05, from Nacalai Tesque, Inc , Kyoto, Japan), by adding Glucose, NaHCO3, and either all 20 AAs (for full medium) or 18–19 AAs only (for deprivations of two-one AAs)  Single AAs, Glucose, and NaHCO3 were from Sigma  Further details and amounts utilized are indicated in S1 Table  All media were supplemented with 10% dialyzed FBS (Invitrogen), 100 U/ml penicillin G (Invitrogen), 100 mg/ml streptomycin (Invitrogen), and G418 as required  HBSS was from Invitrogen  Cells were seeded at 10–30% of confluency; cells to be starved for 48 h were plated 2–3 times more confluent compared to the control  The following day, cells were washed and cultured in the appropriate medium, with or without EAA, for 24–48 h 
L-Histidinol (HisOH), PP242, Integrated Stress Response Inhibitor (ISRIB), SP600125, Cycloheximide (CHX) were from Sigma; Salubrinal was from Tocris Bioscience; U0126 was from Promega  Drugs were used at the following final concentrations: HisOH at 4–16 mM; PP242 at 1–3 μM; ISRIB at 100 nM; SP600125 at 20 μM in HepG2 cells and 50 μM in HeLa cells; Cycloheximide (CHX) at 50 ug/ml in HepG2 cells and 100 ug/ml in HeLa cells; Salubrinal at 75 μM; U0126 at 50 μM  Vehicle was used as mock control  Treatments with drugs to be tested for their ability to inhibit transgene reactivation (ISRIB, SP600125 and U0126) were initiated 1h before the subsequent addition of L-Histidinol (ISRIB) or the subsequent depletion of Met/Cys (SP600125 and U0126) 
Total RNA was purified using the RNeasy Mini kit (Qiagen), according to manufacturer’s instructions  RNA concentration was determined by Nanodrop 8000 Spectrophotometer (Thermo Scientific)  Equal amount (1 μg) of RNA from HeLa, HepG2 and C2C12 cells was reverse transcribed using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) using oligo-dT as primers, and diluted to 5 ng/μl  The cDNA (2 μl) was amplified by real-time PCR using SYBR green Master Mix on a Light Cycler 480 (Roche), according to manufacturer’s instructions  The thermal cycling conditions were: 1 cycle at 95°C for 5 min, followed by 40–45 cycles at 95° for 20 sec, 56° for 20 sec and 72° for 20 sec  The sequences, efficiencies and annealing temperatures of the primers are provided in S2 Table  Data were analyzed with Microsoft Excel using the formula EtargetΔct target (control-sample) /EreferenceΔct reference (control-sample)   Reference genes for normalizations were ARPC2 (actin-related protein 2/3 complex, subunit 2) for HeLa and HepG2 cells; and Actb (actin beta) for C2C12 cells, unless otherwise indicated 
siRNA (Mission esiRNA, 200 ng/μL; Sigma) against ATF4 and GCN2 were designed against the targeted sequences NM_001675 and NM_001013703, respectively  Cells seeded in 6-well plates were transfected with 1 μg of siRNAs and 5 μL of Lipofectamine 2000 (Invitrogen), following manufacturer’s instructions, at day 1 post-plating for ATF4 and at day 1 and 2 post-plating for GCN2  At day 2 (ATF4) or 3 (GCN2) post-plating, cells were washed and cultured in medium in the absence or presence of HisOH 4 mM for 6 h  siRNAs against RLuc (Sigma), targeting Renilla Luciferase, were used as negative control  For CRISPR/Cas9 experiments, we used the “all-in-one Cas9-reporter” vector, expressing GFP (Sigma), which is characterized by a single vector format including the Cas9 protein expression cassette and gRNA (guide RNA)  GFP is co-expressed from the same mRNA as the Cas9 protein, enabling tracking of transfection efficiency and enrichment of transfected cells by fluorescence activated cell sorting (FACS)  The human U6 promoter drives gRNA expression, and the CMV promoter drives Cas9 and GFP expression  The oligonucleotide sequences for the three gRNAs targeting GCN2 exon 1 or 6 are listed in S2 Table  We transfected HeLa and HepG2 cells with these plasmids individually (one plasmid one guide) and sorted the GFP-positive, transfected cells by FACS  Screening GCN2-KO clones was performed by western blotting  In the case of HepG2-OA1 cells, two rounds of selection were necessary to obtain three GCN2-KO clones by using a guide RNA against exon 1  Compared to the original HepG2-OA1 cell line and to the clone resulting from the first round of selection (185#27), the selected clones E23, F22 and F27 showed a very low amount—if any—of residual GCN2 protein (see results) 
Genomic DNA of HeLa and HepG2 cells was purified using DNeasy Blood and Tissue kit (Qiagen), according to the manufacturer’s instructions  DNA concentration was determined by Nanodrop 8000 Spectrophotometer (Thermo Scientific)  PCR conditions for amplification of GCN2 exon 1 and 6 were as follows: 1 cycle at 94°C for 5 min, followed by 35 cycles at 94°C for 40 sec, 56°C for 40 sec, and 72°C for 40 sec; and a final extension step of 5 min at 72°C  The primer sequences are provided in S2 Table 
For OA1, western immunoblotting was carried out as described   For GCN2, cells were lysed in RIPA buffer, boiled at 95°C for 5 min and resolved on a 7 5% polyacrylamide gel; immunoblotting was then performed following standard procedures  Primary Abs were as follows: anti-human OA1, previously developed by our group in rabbits ; anti-GCN2 (Cell Signaling, Cat  #3302) 
Statistical analyses were performed using Microsoft Excel for Mac (version 15 32, Microsoft) for Student’s t-test; or GraphPad Prism (version 5 0d for Mac, GraphPad Software, Inc ) for one-way analysis of variance (ANOVA), followed by Dunnett’s or Tukey’s multiple comparisons post-tests  T-test was used when only two means, typically sample versus control, were compared, as specified in the figure legends  One way ANOVA was used for multiple comparisons, followed by either a Dunnett’s (to compare every mean to a control mean), or a Tukey’s (to compare every mean with every other mean) post-test, by setting the significance level at 0 05 (95% confidence intervals)  Both tests compare the difference between means to the amount of scatter, quantified using information from all the groups  Specifically, Prism computes the Tukey-Kramer test, allowing unequal sample sizes  P values in Figures are generally referred to comparison between a sample and the control (full medium/mock), and are indicated as follows: *P<0 05, **P<0 01, ***P<0 001  Comparisons not involving the control are similarly indicated, by a horizontal line at the top of the graphs, encompassing the two samples under analysis  Additional details regarding the specific experiments are reported in the Figure Legends 
To examine the expression behavior of genomic repeats upon AA starvation, we performed a transcriptomic analysis taking advantage of an intramural sequencing facility  HeLa-OA1 cells were cultured in normal medium (for 6-30-120 hours) or in absence of Met/Cys (for 6-15-30-72-120 hours)  Total RNA was prepared using Trizol (Sigma) to preserve transcripts of both small and long sizes (from Alu, of about 0 3 kb, to Long Interspersed Nuclear Elements, LINEs, and ERVs, up to 6–8 kb long), DNase treated to avoid contamination of genomic DNA, and processed for NGS sequencing by Ovation RNA-Seq System V2 protocol and HiSeq 2000 apparatus  Raw sequence data (10–20 M reads/sample) were aligned to the human genome (build hg19) with SOAPSplice   Read counts over repeated regions, defined by RepeatMasker track from UCSC genome browser , were obtained using bedtools suite   Normalization factors and read dispersion (d) were estimated with edgeR , variation of abundance during time was analyzed using maSigPro package , fitting with a negative binomial distribution (Θ = 1/d, Q = 0 01), with a cutoff on stepwise regression fit r2 = 0 7  Read counts were transformed to RPKM for visualization purposes  The expression of the OA1 transgene and HDAC4, which are progressively up- and down-regulated during starvation, respectively , were used as internal controls 
For genomic repeat analysis, reads belonging to repetitive elements were classified according to RepeatMasker and assigned to repeat classes (total number in the genome = 21), families (total number in the genome = 56) and finally subfamilies (total number in the genome = 1396), each including a variable number of genomic loci (from a few hundred for endogenous retroviruses, up to several thousand for Alu)  Repeat subfamilies were then clustered according to their expression pattern in starved vs control cells, by maSigPro using default parameters, and repeats classes or families that are significantly enriched in each cluster, compared to all genomic repeats, were identified by applying a Fisher Exact test (using scipy stats, a statistical module of Python)  Alternatively, differentially expressed repeat subfamilies were identified by averaging three time points of starvation (15-30-72 h) and controls  Repeats significantly up- or downregulated (104 and 77, respectively) were selected based on a P value <0 05 (unpaired two-tailed Student’s t-test, assuming equal variance), and analyzed for their class enrichment by a Fisher Exact test as described above 
For gene set enrichment analysis of Met/Cys deprived vs control HeLa cells, differentially expressed genes were selected considering three time points of starvation (15-30-72 h) and controls, based on a P value <0 05 (unpaired two-tailed Student’s t-test, assuming equal variance) and a fold change >2  This led to a total of 2033 differentially expressed genes, 996 upregulated and 1037 downregulated  The enrichment analysis was performed separately for up and down regulated genes, or with all differentially expressed genes together (both), using the KEGG database  The analysis was performed with correction for the background of all expressed genes (about 13600 genes showing an average expression over 3 starvation and 3 control samples of at least 5 counts) and by using default parameters (adjusted P value and q-value cut-off of <0 05 and 0 2, respectively)  Differentially expressed genes were also selected considering all starvation time points, as with genomic repeats, by maSigPro using default parameters, and a fold change of at least 1 5, leading to similar enrichment results (not shown)  RNAseq gene expression data are available in the ArrayExpress database under the accession number E-MTAB-6452 
To provide proof-of-principle that AA starvation may affect the expression of transposable elements, we performed an RNAseq analysis of the previously described HeLa-OA1 cells, carrying an integrated and partially silenced OA1 transgene   Since the reactivation of the transgene by starvation is a progressive phenomenon , we performed a time-course experiment, where each time point represents one biological sample, rather than a biological triplicate of a single time point  To this aim, cells were cultured either in normal medium, or in absence of Met/Cys for different time points (6-15-30-72-120 hours), resulting in the progressive upregulation of the OA1 transgene during starvation (Fig 1A and 1B), consistent with previously published results   The expression of genomic repeats was determined according to RepeatMasker annotation and classification into classes, families, and subfamilies  Repeat species were then subjected to differential expression and enrichment analyses in starved vs control conditions  Out of 1396 annotated repeat subfamilies, 172 species displayed a differential expression profile during starvation 
Fig 1



ground_truth: 48V.

prompt_front: Now, answer the following question based on the text below, only give me the answer and do not output any other words.

Question: Is the ISR necessary for transgene reactivation?

Read the following text and answer briefly.

Context 3:  Exogenous transgene and endogenous retroviruses are upregulated in Met/Cys-deprived HeLa cells 
(A,B) Exogenous integrated transgene (OA1) mRNA abundance in HeLa-OA1 cells, cultured in Met/Cys-deprived medium for the indicated time points, and analyzed by RNAseq (A), or RT-qPCR (B), compared to full medium  Data represent RPKM (A), or mean ± SD of 2 technical replicates, expressed as fold change vs  control (full medium at 6 h = 1) (B)  (C) Clustering of 172 genomic repeat subfamilies, differentially expressed upon starvation, according to their expression profile  (D) Class distribution of repeat subfamilies belonging to differential expression clusters, compared to all genomic repeat subfamilies (first column)  Class DNA includes DNA transposons; SINE includes Alu; LINE includes L1 an L2; LTR includes endogenous retroviruses and solitary LTRs; Satellite includes centromeric acrosomal and telomeric satellites; Others includes SVA, simple repeats, snRNA, and tRNAs  LTR-retroelements are significantly enriched among repeats that are upregulated upon starvation, while LINEs are significantly enriched among repeats that are downregulated  *P<0 05, ***P<0 001 (Fisher exact test) 
As shown in Fig 1C, the clustering of differentially expressed repeats, according to their expression pattern, reveals profiles comparable to the behavior of the transgene in the same conditions, i e  upregulation upon starvation and no change in regular medium (Cluster 1 and 2)  In particular, Cluster 1 contains sequences that, similarly to the OA1 transgene, are progressively upregulated upon starvation (Fig 1A and 1C) , while Cluster 2 contains sequences that are upregulated at early time points  Interestingly, repeat families that are significantly enriched in these two clusters belong mostly to the group of LTR-retrotransposons, including ERV1, ERVK, ERVL, ERVL-MaLR and other LTR sequences (Fig 1D; S1A and S2A Figs)  By contrast, DNA transposons (such as TcMar-Tigger) and L1 non-LTR retrotransposons are enriched among repeats that are downregulated during starvation, particularly at late time points (Clusters 3 and 4) (Fig 1D; S1A and S2A Figs)  Consistent results were obtained by selecting significantly up- or downregulated genomic repeats (overall 181 species), based on their average expression out of three time points of starvation (15-30-72 h, when the transgene upregulation is more homogeneous) and controls, and on a P value <0 05 (S1B and S2B Figs)  These findings suggest that EAA starvation induces genome-wide effects involving repetitive elements, and that—among major repeat classes—it upregulates in particular the expression of ERVs 
In addition, to obtain a general overview of main gene pathways changing their expression together with the transgene during AA starvation, we performed gene expression and enrichment analyses of regular genes, by considering three time points of starvation (15-30-72 h) and controls  Differentially expressed genes were selected based on a P value <0 05 and a fold change between means of at least 2, and analyzed with the EnrichR tool   As shown in Fig 2 and S1 File, enrichment analyses against the KEGG and Reactome databases reveals a predominance of downregulated pathways, namely ribosome and translation, proteasome, AA metabolism, oxidative phosphorylation and other pathways related to mitochondrial functions, which are affected in Huntington, Alzheimer and Parkinson diseases (http://www genome jp/kegg/pathway html)  In particular, a large fraction of ribosomal protein mRNAs is downregulated upon Met/Cys starvation (Fig 2A and 2C; S1 File), consistent with the notion that their genes–despite being scattered throughout the genome—are coordinately expressed in a variety of conditions   This reduced expression may depend on multiple pathways that control ribosome biogenesis in response to external stimuli, including the downregulation of Myc activity , the downregulation of mTORC1 [42, 44], or possibly the activation of the ISR, as described in yeast   By contrast, upregulated genes show a significant enrichment for transcription and gene expression (Fig 2B)  Similar results were obtained by the Gene Ontology Biological Process (GO-BP) database (S1 File), overall indicating a general downregulation of translation and metabolism, and upregulation of transcription, during the time interval of Met/Cys starvation corresponding to the transgene upregulation 
Fig 2  Gene set enrichment analysis of Met/Cys-deprived HeLa cells 
Differentially expressed genes between three time points of starvation (15-30-72 h) and controls were selected based on a P value <0 05 and a fold change of at least 2, leading to a total of 996 upregulated, and 1037 downregulated genes  The enrichment analysis was performed separately for up and down regulated genes, using the EnrichR tool and the KEGG (A) and REACTOME (B, C) databases  Ranking is based on the combined score provided by EnrichR, and categories are displayed up to 20 items with an Adjusted P value <0 05  No significant categories were found with upregulated genes against the KEGG database  All data are shown in S1 File  The enrichment analysis using all differentially expressed genes together did not reveal any additional enriched process 
To characterize the pathway leading to the reactivation of silenced transgenes, we used HeLa-OA1 and HeLa-GFP cells, as described   In addition, to test cell types relevant for AA metabolism, such as liver and muscle, we generated clones of HepG2 human hepatoma and C2C12 mouse skeletal muscle cells, stably transfected with plasmids for OA1 and GFP transgenes, respectively (HepG2-OA1 and C2C12-GFP cells; endogenous OA1 is not expressed in any of these cell types)  In all cases, the integrated transgenes are under the control of the CMV promoter in the context of a pcDNA3 1 plasmid, are partially silenced, and can be efficiently upregulated by HDAC inhibitors (trichostatin A, TSA; ref   and S3A, S3B and S4A Figs), indicating that their expression is controlled at least in part by epigenetic mechanisms, as previously described  
To establish whether the reactivation response results from the shortage of specific AAs only, such as Met/Cys, or it is triggered by any AA deprivations, we cultured HeLa-OA1, HeLa-GFP, HepG2-OA1 and C2C12-GFP cells for 24–48 hours with a battery of media deprived of EAAs or semi-EAAs, including Met/Cys, Thr, Gln, Val, Leu, Tyr, Trp, Lys, and His  As negative controls, cells were cultured in full medium, carrying the entire AA complement, and in a medium deprived of Ala, a non-essential AA  The expression of the transgene transcript was then evaluated by RT-qPCR  As shown in Fig 3, and in S3C and S4B Figs, most EAA-deficiencies induced reactivation of the OA1 or GFP transgenes in all four cell lines, with the notable exception of Trp deprivation, which consistently resulted in no or minimal reactivation of the transgenes  Indeed, despite some variability, Met/Cys deficiency, but also Thr, Val, Tyr, and His deprivation always gave an efficient response, while Leu, Gln and Lys elicited evident responses in some cases, but not in others  Depletion of Phe gave results comparable to Tyr deprivation, however it significantly altered multiple reference genes used for normalization and therefore was eventually omitted from the analysis (not shown)  Finally, in the above experiments we used a combined Met/Cys deficiency, to avoid the potential sparing of Met by Cys  and for consistency with our previous studies   Nevertheless, the analysis of single Met or Cys starvation, both at the protein and transcript levels, revealed an exclusive role of Met deprivation in transgene reactivation, consistent with the notion that Cys is not an EAA (S3D and S3E Fig) 
Fig 3  EAA deprivation induces reactivation of silent transgenes in HeLa and HepG2 cells 
Relative transgene (OA1) and CHOP mRNA abundance in HeLa-OA1 (A) and HepG2-OA1 (B) cells, cultured in various AA-deprived media for 48 h and 24 h, respectively, compared to full medium  Mean ± SEM of 3 independent experiments  Data are expressed as fold change vs  control (full medium = 1)  *P<0 05, **P<0 01, ***P<0 001 (one way ANOVA, followed by Dunnett’s post-test vs  full medium) 
Collectively, these results indicate that transgene reactivation by EAA starvation is reproducible with most EAAs, shared by different cell types (epithelium, liver, and skeletal muscle), and conserved in different mammalian species (human, mouse) 
mTORC1 inhibition and GCN2 activation trigger the best-known signaling pathways responding to AA starvation   We previously showed that inhibition of mTORC1 is not sufficient to reproduce transgene reactivation in HeLa cells   By contrast, the involvement of GCN2 and the ISR, including the downstream effectors ATF4 and CHOP, has never been tested  In addition, this pathway has been typically assessed in transient assays, lasting for a few hours, which may not be comparable with the prolonged starvation conditions necessary to reactivate the transgene expression (at least 15–24 h)  Thus, we tested whether CHOP expression was upregulated upon incubation of HeLa-OA1, HepG2-OA1 and C2C12-GFP cells in media deprived of different EAAs for 24–48 h 
As shown in Fig 3 and S4B Fig, we found that CHOP expression is increased in all EAA-starvation conditions, but not in the absence of Ala, in all tested cell lines  Similar, yet less pronounced, results were obtained with ATF4, consistent with the notion that activation of this transcription factor is mainly mediated by translational upregulation (not shown) [15, 26]  However, the upregulation of CHOP does not parallel quantitatively that of the transgene, neither appears sufficient to induce it  In fact, CHOP is highly upregulated even upon Trp starvation, which consistently results in no or minimal reactivation of the transgenes (compare CHOP with OA1 or GFP expression; Fig 3 and S4B Fig)  Thus, while the ISR appears widely activated upon EAA starvation, the upregulation of its downstream effector CHOP only partly correlates with transgene reactivation and may not be sufficient to induce it 
The activation of the ISR upon AA starvation suggests that GCN2 may be involved in the transgene reactivation response  Therefore, we tested whether direct pharmacological activation of this kinase is sufficient to trigger the transgene reactivation similarly to starvation  In addition, we used pharmacological inhibitors of mTOR to corroborate previous negative results in HeLa cells  in the other cell lines under study  To this aim, HeLa-OA1 or GFP, HepG2-OA1 and C2C12-GFP cells were cultured in the presence of different concentrations of PP242 (mTOR inhibitor) or L-Histidinol (GCN2 activator, inhibiting tRNAHis charging by histidyl-tRNA synthetase), either alone or in combination for 24 h, compared to Met/Cys-deprived and full medium  As shown in Fig 4 and S5 Fig, while inhibition of mTORC1 consistently leads to minor or no effects, in agreement with previous findings , treatment with L-Histidinol results in efficient reactivation of the transgene in HepG2-OA1 and C2C12-GFP cells, but not in HeLa cells 
Fig 4  mTOR inhibition and GCN2 activation differently affect transgene expression in HeLa and HepG2 cells 
Relative transgene (OA1) and CHOP mRNA abundance in HeLa-OA1 (A) and HepG2-OA1 (B) cells, cultured in Met/Cys-deprived medium, or in the presence of PP242 (mTOR inhibitor; 1–3 μM) or L-Histidinol (HisOH, GCN2 activator; 4–16 mM), either alone or in combination for 24–48 h, compared to full medium  Mean ± SEM of 4 (A) or 3 (B) independent experiments  Data are expressed as fold change vs  control (full medium = 1)  *P<0 05, **P<0 01, ***P<0 001 (one way ANOVA, followed by Dunnett’s post-test vs  full medium)  PP-1 and PP-3, PP242 at 1 and 3 μM, respectively; HisOH-4 and HisOH-16, L-Histidinol at 4 and 16 mM, respectively 
Specifically, L-Histidinol is not effective in HeLa-OA1 and HeLa-GFP cells, either alone or in combination with PP242 (Fig 4A and S5A Fig), or by using different concentrations of the drug, with or without serum (not shown)  In these cells, L-Histidinol appears also unable to trigger the ISR, as indicated by lack of CHOP upregulation, possibly due to their different sensitivity to the drug  These findings are consistent with previous reports, describing the use of L-Histidinol in HeLa cells in conditions of low His concentration in the culture medium , which would resemble AA starvation in our system and therefore may not be applicable  Thus, even though the amount of the amino alcohol was adapted to exceed 20 to 80 times that of the amino acid, as described , HeLa cells may be resistant or able to compensate 
In contrast, in other cell types, L-Histidinol has been utilized in regular DMEM, to mimic the AA response triggered by DMEM lacking His [48, 49]  Consistently, in HepG2-OA1 cells, L-Histidinol is sufficient to elicit extremely high levels of transgene reactivation, and its combination with PP242 results in additive or even synergistic effects, possibly due to an indirect effect of mTOR inhibition on GCN2 activity (Fig 4B) [50, 51]  Similarly, C2C12-GFP cells efficiently reactivate the transgene upon treatment with L-Histidinol, but not PP242 (S5B Fig)  However, differently from HepG2-OA1 cells, simultaneous treatment of C2C12-GFP cells with L-Histidinol and PP242 does not lead to synergistic effects  Consistent with stimulation of the ISR, CHOP and to a minor extent ATF4 are upregulated by L-Histidinol in both cell lines, yet their expression levels show only an incomplete correlation with those of the transgene (Fig 4B, S5B Fig, and not shown) 
The finding that GCN2 activation by L-Histidinol is sufficient to reactivate the transgenes in both HepG2-OA1 and C2C12-GFP cells pointed to this kinase, and to the downstream ISR, as the pathway possibly involved in the EAA starvation response  Thus, we investigated whether the ISR is sufficient to trigger upregulation of the OA1 transgene in HepG2-OA1 cells by pharmacological means  As CHOP expression does not correspond quantitatively and is not sufficient to induce transgene reactivation, we tested the role of the core upstream event of the ISR, namely the phosphorylation of eIF2α , which can be induced by pharmacological treatments, independent of GCN2 (Fig 5A)  To this aim, we used Salubrinal, a specific phosphatase inhibitor that blocks both constitutive and ER stress-induced phosphatase complexes against eIF2α, thereby increasing its phosphorylation   We found that, while the ISR is activated upon Salubrinal treatment, as shown by increased CHOP expression, it does not induce OA1 transgene reactivation (Fig 5B) 
Fig 5  The ISR is neither sufficient nor necessary to induce transgene reactivation in HepG2 cells 
(A) Schematic representation of GCN2 activation by AA starvation, resulting in phosphorylation of eIF2a and initiation of the downstream ISR  In addition to GCN2, the ISR may be activated by other eIF2a kinases (PKR, HRI and PERK; not shown in the picture)  (B) Relative transgene (OA1) and CHOP mRNA abundance in HepG2-OA1 cells treated for 24 h with Salubrinal (a drug that induces the ISR by inhibiting the dephosphorylation of eIF2α; 75 μM), compared to full medium  Mean ± range of two experiments  Data are expressed as fold change vs  control (DMEM = 1)  *P<0 05 (paired two-tailed Student’s t-test vs  control)  (C) Relative transgene (OA1) and CHOP mRNA abundance in HepG2-OA1 cells treated for 6 h with L-Histidinol (HisOH, GCN2 activator; 4 mM), in the absence or presence of ISRIB (a drug that bypasses the phosphorylation of eIF2α, inhibiting triggering of the ISR; 100 nM)  Mean ± range of two experiments  Data are expressed as fold change vs  control (DMEM = 1)  **P<0 01, ***P<0 001 (one way ANOVA, followed by Tukey’s post-test; P values refer to comparisons vs  control, unless otherwise indicated)  (D) Relative transgene (OA1) and ATF4 mRNA abundance in HepG2-OA1 cells transfected with control (CTRL) or anti-ATF4 siRNAs, and incubated in the presence or absence of L-Histidinol (HisOH, GCN2 activator; 4 mM) for 6 h  Mean ± range of two experiments  Data are expressed as fold change vs  control (w/o HisOH = 1, top; control siRNA = 1, bottom)  *P<0 05 (one way ANOVA, followed by Tukey’s post-test; P values refer to comparisons vs  control, unless otherwise indicated) 
To test whether the ISR is necessary to trigger the transgene response to L-Histidinol, we used the chemical compound ISRIB, which inhibits the activation of the ISR, even in the presence of phosphorylated eIF2α, likely by boosting the activity of the guanine-nucleotide exchange factor (GEF) for eIF2α, namely eIF2B [53, 54]  HepG2-OA1 cells were stimulated with L-Histidinol, either in the presence or absence of ISRIB  As shown in Fig 5C, while the expression of CHOP is inhibited by ISRIB, as expected, the reactivation of the OA1 transgene is not affected  In addition, knockdown of the closest eIF2α downstream effector ATF4 by siRNAs does not interfere with the reactivation of the OA1 transgene by L-Histidinol (Fig 5D)  Together, these data suggest that eIF2α phosphorylation and the downstream ISR pathway are neither sufficient nor necessary to induce transgene reactivation 
To definitively establish if GCN2 is necessary to trigger the transgene reactivation response to EAA starvation, we directly suppressed its expression by CRISPR/Cas9-mediated knock-out (KO)  We generated three independent GCN2-KO clones from the parental HeLa-OA1 cell line, by using three different guide RNAs, two against exon 1 (clones 183#11 and 185#5), and one against exon 6 (clone 239#1) of the GCN2 gene  Genomic characterization confirmed the presence of mutations on both alleles of exon 1 of the GCN2 gene in clone 183#11, and on both alleles of exon 6 in clone 239#1; by contrast, clone 185#5 showed multiple alleles in exon 1, consistent with the presence of two cell populations, and was not characterized further at the genomic level (S6 Fig)  None of these clones express GCN2 at the protein level, as shown by immunoblotting (Fig 6A)  To test the GCN2-KO cells for their ability to respond to EAA starvation, parental HeLa-OA1 cells and the three GCN2-KO clones were cultured in media deprived of Met/Cys or Thr (corresponding to the most effective treatments in this cell line; see Fig 3A) for 24–48 h and transgene expression was assessed by RT-qPCR  We found that the reactivation of the OA1 transgene is neither abolished, nor reduced by KO of GCN2, thus excluding that this kinase is necessary for the response to EAA starvation in HeLa-OA1 cells (Fig 6B and 6C) 
Fig 6  GCN2 knockout does not interfere with transgene reactivation in HeLa cells 
(A) Immunoblotting of protein extracts from the HeLa-OA1 parental cell line and GCN2-KO clones 183#11, 185#5 and 239#1, immunodecorated with anti-GCN2 antibody  Arrow, GCN2 specific band  Ponceau staining was used as loading control  (B, C) Relative transgene (OA1) mRNA abundance in HeLa-OA1 cells and GCN2-KO clones, cultured in Met/Cys (B) or Thr (C) deprived medium for 24 h or 48 h, respectively, compared to full medium  Mean ± SD of 3 technical replicates from 1 experiment  Data are expressed as fold change vs  control (full medium = 1)  Since independent clones may display variable reactivation responses (e g  due to different levels of transgene expression in basal conditions), the results are not shown as means of the three clones, but as separate replicates 
Similarly, we generated GCN2-KO clones from the parental HepG2-OA1 cell line by the same strategy  By using a guide RNA against exon 1 of the GCN2 gene, we obtained three independent GCN2-KO clones, namely E23, F22 and F27  Genomic characterization confirmed the presence of mutations on both alleles of exon 1 of the GCN2 gene in clone F27 (S7 Fig) and all three clones showed a very low amount—if any—of residual GCN2 protein, compared to the original HepG2-OA1 cell line (Fig 7A)  To assess the ability of GCN2-KO cells to reactivate the transgene upon starvation, we cultured parental HepG2-OA1 cells and the three GCN2-KO clones in media deprived of Met/Cys or His (corresponding to the most effective treatments in this cell line; see Fig 3B) for 24 h, and evaluated the transgene expression by RT-qPCR  As shown in Fig 7B and 7C, we found that the reactivation of the OA1 transgene is neither abolished, nor reduced by KO of GCN2, as in HeLa cells  To further confirm this result, we knocked-down GCN2 by RNA interference (RNAi), and incubated the cells with or without L-Histidinol for 6 h  As shown in Fig 8, treatment of HepG2-OA1 cells with L-Histidinol results in efficient transgene reactivation, even upon significant GCN2 downregulation, both at the mRNA and protein levels  Taken together, these data strongly support the conclusion that GCN2 is not necessary for transgene reactivation in response to EAA starvation, either in HeLa or in HepG2 cells 
Fig 7  GCN2 knockout does not interfere with transgene reactivation in HepG2 cells 
(A) Immunoblotting of protein extracts from the HepG2-OA1 parental cell line and GCN2-KO clones 185#27, E23, F22, F27, immunodecorated with anti-GCN2 antibody  Clone 185#27 results from the first round of selection, and was used to generate clones E23, F22, F27  Arrow, GCN2 specific band  For GCN2 protein quantification, Ponceau staining was used as loading control and data are expressed as fold change vs  parental cell line (= 1)  (B, C) Relative transgene (OA1) mRNA abundance in HepG2-OA1 cells and GCN2-KO clones, cultured in Met/Cys (B) or His (C) deprived medium for 24 h, compared to full medium  Mean ± SD of 3 technical replicates from 1 experiment 



ground_truth: No, it is not necessary.