PEComas: the past, the present and the future

The perivascular epithelioid cell (PEC) is a cell type constantly present in a group of tumors called PEComas. PEC expresses myogenic and melanocytic markers, such as HMB45 and actin. Recently, recurrent chromosomal alterations have been demonstrated in PEC. At present, PEComa is a widely accepted entity. In the past 10 years, the use of this term has allowed to report and describe numerous cases permitting to start highlighting the biology of this group of lesions. PEComas are related to the genetic alterations of tuberous sclerosis complex (TSC), an autosomal dominant genetic disease due to losses of TSC1 (9q34) or TSC2 (16p13.3) genes which seem to have a role in the regulation of the Rheb/mTOR/p70S6K pathway. There are some open questions about PEComas regarding its histogenesis, the definition of epithelioid angiomyolipoma and the identification of the histological criteria of malignancy. An innovative therapeutic trial using rapamycin is under way for tumors occurring in TSC such as renal angiomyolipoma and lymphangioleiomyomatosis. Its success could provide the rationale for the use of the same drug in other lesions composed of PECs, especially in the malignant ones.
What is the perivascular epithelioid cell?
The perivascular epithelioid cell (PEC) (Fig. 1) is a cell type constantly present in a group of tumors including angiomyolipoma (AML), clear-cell “sugar” tumor (CCST) of the lung and extrapulmonary sites, lymphangioleiomyomatosis, clear-cell myomelanocytic tumor of the falciform ligament/ligamentum teres and rare clear-cell tumors of other anatomical sites.
Fig. 1Renal angiomyolipoma: perivascular epithelioid cells arranged around a blood vessel; H&E ×20
It has morphologic, immunohistochemical, ultrastructural and genetic distinctive features such as an epithelioid appearance with a clear to granular cytoplasm, a round to oval, centrally located nucleus and an inconspicuous nucleolus. PEC has mild to any atypia and a typical perivascular location [13]. At present, PEC has not a known normal counterpart.
Immunohistochemically, PEC expresses myogenic and melanocytic markers, such as HMB45, HMSA-1, MelanA/Mart1, microophtalmia transcription factor (Mitf), actin and, less commonly, desmin [13, 55, 126]. Its immunoreactivity for vimentin is usually incospicuous.
At ultrastructural analysis, PEC contains microfilament bundles with electron-dense condensation, numerous mithocondria and membrane-bound dense granules [11, 118].
It is thought that PEC can modulate its morphology and immunophenotype: given all the characteristics described above (Fig. 2), PEC can show muscular features with a spindle shape and a stronger positivity for actin than for HMB45 or it can have an epithelioid feature with a strong positivity for HMB45 and a mild, if any, reaction for actin (Fig. 3). PEC can also become vacuolized acquiring the feature of an adipocyte. Progesterone receptor positivity has been described in PEC with spindle morphology; this suggests a possible role of progesterone in this morphologic modulation [13, 107].
Fig. 2Diagram demonstrating the modulation of morphology and immunophenotye of PECsFig. 3Hepatic angiomyolipoma: strong granular HMB45 immunoreactivity in perivascular epithelioid cells; HMB45 ×20
Recently, recurrent chromosomal alterations have been demonstrated in PEC [87].
What is a PEComa?
The World Health Organization defines PEComa as “a mesenchymal tumor composed of histologically and immunohistochemically distinctive perivascular epithelioid cells” [37].
At present, this neoplasm is a widely accepted entity. However, some authors cast doubts on the existence of PEComa as a distinctive tumor, particularly as regards the uterine ones that they view as leiomyosarcoma with aberrant expression of HMB45 [102–104]. Although we understand their perplexity, we would like to underline some points. First, it has been demonstrated that there are different gene expression profiles among uterine smooth muscle tumors, demonstrating their heterogeneity [105, 106, 119]. Second, we think that it is difficult to explain, in uterine smooth muscle neoplasms, both HMB45 and Mitf positivity as an aberrant expression or antibody crossreactivity involving both antigens [81]. Third, we think that the absence of a recognized normal counterpart for PEC is not a sufficient reason to reject the concept of PEComa; in fact, there are other well-accepted soft-tissue neoplasms, such as alveolar soft-part sarcoma and epithelioid sarcoma, that still have not a known normal counterpart.
Some authors have questioned about the use of the term “PEComa”. They consider it ambiguous because it is not clear whether this term should be restricted to purely epithelioid tumors as CCST or whether the term should be more broadly applied to include AML and lymphangioleiomyomatosis [80, 111].
We believe that AML, CCST and pulmonary lymphangioleiomyomatosis are composed of PECs in different stages of modulation, and these lesions, together with clear-cell myomelanocytic tumor of the falciform ligament/ligamentum teres, belong to the same family of tumors, the PEComas.
Nevertheless, we think that it is better to continue to name some of them (i.e. AML and pulmonary lymphangioleiomyomatosis) also with their established names which identify their clinical and morphological aspects and are well known by clinicians and pathologists [15].
Moreover, in the past 10 years, the use of the term PEComa has permitted to report and describe numerous cases with the morphological and immunohistochemical features of this tumor permitting to start to understand the biology of this group of lesions.
PEC and tuberous sclerosis
PEComas are related to the tuberous sclerosis complex (TSC) or, better, to the genetic alterations of TSC, an autosomal dominant genetic disease due to losses of TSC1 (9q34) or TSC2 (16p13.3) genes [112, 116] and characterized by mental retardation, seizures and cellular proliferations (AMLs, subependymal giant cell tumors, cutaneous angiofibromas, cardiac rabdomyomas, lymphangioleiomyomatosis, pulmonary multifocal micronodular hyperplasia). Similar alterations of the TSC genes have been demonstrated in a significant number of PEComas, both occurring within the TSC and in sporadic cases.
In recent years, great advances have been made in our knowledge of TSC and related lesions. In particular, TSC genes seem to have an important role in the regulation of the Rheb/mTOR/p70S6K pathway [62].
Kenerson et al. [59] have recently demonstrated increased levels of phospho-p70S6K, a marker of mTOR activity, in sporadic AMLs. The associated reduced phospho-AKT expression is consistent with the disruption of TSC1/2 function.
Similar findings were obtained analysing extrarenal PEComas.
PEComa: the past
PEC was first described in 1943 by Apitz [5] as an “abnormal myoblast” in renal AML, as reported by Masson [77] in his famous book.
The idea of a possible link between AML and other two lesions, CCST and lymphangioleiomyomatosis, came from the fact that the same epithelioid cell was observed in them. Moreover, this cell was HMB45-positive in all three lesions [9, 93, 94].
Later, ultrastructural examination revealed the presence of premelanosomes both in CCSTs of the lung and in epithelioid clear-cell component of renal and hepatic AMLs [9, 42, 43, 93, 94, 118].
In addition, the possibility of a relationship between AML and lymphangioleiomyomatosis was suggested by the fact that both occur in TSC [10].
We proposed that CCST should be also included in this syndrome [10], and the first description of this tumor in a patient with TSC confirmed this idea [35].
In 1996, we described the first case of pancreatic CCST and suggested the name PEComa for neoplasms composed of a pure proliferation of PECs [125].
Moreover, the fact that the same clear epithelioid cell composing pulmonary “sugar” tumor was present in AML and was the only component of some small renal microhamartoma (Fig. 4) suggested to us the idea of the existence of a monotypic epithelioid AML of the kidney. We looked for them, and we found them [96].
Fig. 4Renal microhamartoma completely composed of perivascular epithelioid cells; H&E ×20
PEComa: the present
PEComas have been described in different organs and are considered ubiquitous tumors.
Kidney
PEComas of the kidney include classic AML, microscopic AML (so-called microhamartoma), intraglomerular lesions, cystic AML, epithelioid AML, oncocytoma-like AML and lymphangiomyomatosis of the renal sinus.
Classic angiomyolipoma is the most common mesenchymal tumor of the kidney. Being composed of a variable mixture of adipose tissue and spindle and epithelioid smooth muscle cells mixed together with abnormal thick-walled blood vessels, AML is the prototype of the capacity of PEC to modulate its morphology [29, 76].
For a long time, AML has been considered a hamartoma rather than a true neoplasm, but, at present, its clonal nature has been demonstrated [19, 57, 101]. In patients with TSC, renal AMLs are found in both sexes, in the third and fourth decades of life, with a female predominance; they are usually asymptomatic, bilateral, small and multifocal. Sporadic AMLs occur in older patients, in the fourth to sixth decades of life, with a female predominance; they are single, unilateral and larger than those associated with TSC [76]. Classic AML contains more than one cell type; if a particular cell type predominates, AML is consequently named (lipoma-like AML or leiomyoma-like AML) [6].
Classic AML has a benign outcome. Multifocality and regional lymph node involvement can occur, and this is considered to represent a multifocal growth pattern rather than a metastasis [1, 110]. Three cases of sarcoma developing in sporadic AML have been reported. Similar cases have not been described in TSC patients [22, 32, 73].
Both inherited and sporadic AML frequently demonstrates loss of heterozigosity of chromosome 16p (containing the TSC2 locus). The TSC1 gene occasionally shows loss of heterozigosity [17, 50].
Microscopic angiomyolipomas (so-called microhamartomas) They are small nodules often present in a kidney with an AML. They are not homogeneous in appearance and display all the heterogeneous morphologic aspects of AML; microscopic AMLs do not contain thick-walled blood vessels [13, 20].
Intraglomerular lesions with similar features of AML have been reported in patients with and without tuberous sclerosis and in the TSC2/PKD1 contiguous gene syndrome, a disease with a deletion disrupting both TSC2 and PKD1 (autosomal dominant polycystic disease gene) [74].
Cystic angiomyolipoma is a recently described variant of AML [25, 33]. It is a solid-cystic lesion that, microscopically, is composed of epithelial cysts lined by cuboidal to hobnail cells, positive for cytokeratin; a compact subepithelial “cambium-like” layer of stromal cells positive for HMB45, Melan-A, CD10, estrogen and progesterone receptors and a solid extracystic component with the morphology of a muscle-predominant (Fig. 5) AML, which is positive for HMB45, estrogen and progesterone receptors, smooth muscle actin and desmin and which is associated with irregular blood vessels. Some authors think that the subepithelial “cambium-like” layer is a manifestation of a mullerian differentiation of PEC. If the epithelial cysts represent entrapped renal tubular epithelium [33] or are also related to PEC is unknown.
Fig. 5Cystic angiomyolipoma: strong positivity for actin in the solid extracystic component; SM ACT ×10Only 1 of the 16 cases described to date had a history of TSC [33].
Epithelioid angiomyolipoma is another recently described variant of AML. It is composed of purely epithelioid cells with melanogenesis markers immunoreactivity arranged in sheets. Both adipocytes and abnormal blood vessels are not present. The cytoplasm of the neoplastic cells varies from faintly eosinophilic to clear. Tumor cells can display considerable nuclear atypia, and necrosis can be present. This tumor can recur locally and metastasise causing death. On the basis of histology alone, it is not possible to predict malignant behaviour in these neoplasms, and further data are needed to better define it. However, at the present time, all epithelioid AMLs should be closely followed clinically. Epithelioid AMLs has been described in patient with or without evidence of TSC and in the TSC2/PKD1 contiguous gene syndrome.Loss of heterozigosity of TSC2 have been reported in some cases of sporadic epithelioid AML [28, 72, 74, 84, 96].
Oncocytoma-like angiomyolipomas are tumors composed of a homogeneous population of HMB45-positive polygonal cells with strongly eosinophilic cytoplasm. They have been described in patients with and without TSC. Recognition of this variant is important because oncocytomas in the same kidney with AMLs have been reported repeatedly and in patients with TSC oncocytomas seem to occur more frequently than in general population [75].
Lymphangiomyomatosis of the renal sinus is a plaque-like mass in the wall of the renal pelvis. All three cases reported to date also had renal AMLs, but in only one of them careful postmortem examination of the lungs revealed pulmonary lymphangiomyomatosis [74, 78].
Bladder and prostate
In 2003, Pan et al. [85, 86] have reported two PEComas of the genitourinary tract in patients without TSC. Both tumors were composed of a variable percentage of epithelioid and spindle cells with clear to granular cytoplasm arranged in nests separated by a vascular stroma. Neoplastic cells were positive for HMB45 but not for epithelial markers, vimentin and S100 protein. The prostatic tumor (Fig. 6) showed a low mitotic activity, coagulative necrosis and a malignant behaviour, whereas the neoplasm of the bladder (Fig. 7), lacking these histologic findings, behaved in a benign fashion. Another case of primary PEComa of the bladder has recently been described; also in this case, the patient is well and alive at 48 months after surgery [90].
Fig. 6Prostatic PEComa composed of epithelioid cells with clear cytoplasm arranged in nests; H&E ×4Fig. 7Bladder PEComa composed of epithelioid cells with clear cytoplasm arranged in nests, H&E ×4
Finally, Weinreb et al. [120] have recently described a case of PEComa of the urachal cyst composed of pleomorphic cells and containing necrosis and a high mitotic activity. In this case, they also found a peculiar aspect that they named “pecosis”: remote from the tumor, there were isolated capillaries surrounded by a single layer of clear cells.
Uterus
The first case of PEC tumor of the uterus was reported by Pea et al. [95]: it was a polypoid neoplasm involving the endometrium, which showed morphological features overlapping those of the CCST of the lung (Fig. 8).
Fig. 8Uterine PEComa composed of epithelioid cells with a clear cytoplasm and well-defined cell borders; H&E ×10
Vang and Kempson [115] described eight cases of uterine PEC tumor (“PEComa”). They distinguished a morphologic spectrum of neoplasms varying from tumors with a tongue-like growth pattern composed of sheets of HMB45-positive clear epithelioid cells, which they called group A, to circumscribed tumors composed of hyalinized stroma and neoplastic cells focally positive for HMB45 and extensively immunoreactive for actin and desmin, which they refer to as group B. In two of these eight cases, pelvic lymph nodes contained lymphangiomyomatosis.
Lesions considered to be uterine involvement of lymphangioleiomyomatosis are usually asymptomatic, and some of them correspond to an incidental finding in patients with TSC.
PEComas of the uterus have usually shown benign behaviour, but 13 tumors, 2 of them associated with TSC, were aggressive [16]. In two of six uterine PEComas described by Folpe et al. [39], patients have metastases and one of six patients died of PEComa.
Fadare et al. [31] described a case of uterine PEComa in a patient with TSC in which there was intra-abdominal “PEComatosis”: surgeon found small foci of epithelioid cells in the lamina propria of the small intestine, myometrium and ovarian hilum.
Ovary, vulva and vagina
A tumor with a strong and diffuse HMB45 expression morphologically corresponding to an epithelioid angiomyolipoma has been reported in the ovary [4].
Tazelaar et al. [111] reported a case described as primary extrapulmonary clear-cell “sugar” tumor of the vulva.
Finally, cases of vaginal PEComa have also been described [39, 83].
Lung
PEComas of the lung include lymphangioleiomyomatosis and clear-cell “sugar” tumor.
Lymphangioleiomyomatosis (LAM) (Fig. 9) is a rare and progressive disease that affects the lungs of women, usually in premenopausal age; occasionally, it can be extrapulmonary (as described above). It consists of a nodular, often widespread and bilateral interstitial proliferation of HMB45, actin and desmin-positive smooth muscle cells which can vary from small spindle-shaped cells to large epithelioid cells, usually arranged around thin-walled, branching vascular channels; this proliferation is associated with dilated lymphatics and cystic changes [9, 11, 18, 113].
Fig. 9Pulmonary lymphangioleiomyomatosis composed of epithelioid cells arranged around a vascular channel; H&E ×20
LAM is usually sporadic; patients with TSC are frequently afflicted.
In many cases of LAM, there is a slow progression to pulmonary failure, and the only therapy is lung transplantation. In no-transplanted patient, there is a median survival of 8–10 years [113].
CCST (Fig. 10) was originally described in the lung [68]. It is a rare and benign neoplasm composed of a uniform population of round-to-polygonal epithelioid cells, with a clear or eosinophilic cytoplasm and well-defined cell borders. Tumor cells are surrounded by prominent and thin-walled vascular channels. CCST has a nested or alveolar appearance. We have also observed adipocytic cells in a few cases of CCST [12]. Tumor cells are positive for HMB45 (Fig. 11) [12, 41, 93].
Fig. 10Pulmonary clear-cell “sugar” tumor composed of epithelioid cells with a clear cytoplasm and well-defined cell borders. A prominent and vascular channel is present; H&E ×10Fig. 11Pulmonary clear-cell “sugar” tumor: HMB45 immunoreactivity in tumor cells; HMB45 ×20
Pulmonary CCST is rarely associated with TSC [35]; in the vast majority of cases, it is a sporadic lesion.
Pancreas
PEComa of the pancreas was reported for the first time in 1996 by Zamboni et al. [125]. It was actually the first time that the very term PEC was introduced (Fig. 12).
Fig. 12Pancreatic clear-cell “sugar” tumor: this tumor has overlapping features of the clear-cell “sugar” tumor of the lung: epithelioid cells, with a clear cytoplasm and with a nested or appearance; H&E ×4
The paper reported a tumor with overlapping features of the “benign clear-cell sugar tumor of the lung”. This finding was in our opinion consistent with the hypothesis that similar tumors could possibly arise in many if not all locations.
Thus the term PEComa was introduced to include all similar lesions arising outside the lung. At the time, we believed this to be a unique case. However, in the following years we have observed other two cases of PEComa of the pancreas with overlapping morphological, phenotypical and clinical features.
Thus, while still a very rare disease of the pancreas, PEComa is not an extraordinary finding in this location.
The clinical aspects of PEComa of the pancreas are interesting because they are discovered incidentally during echography of the abdomen and then investigated usually with cyto-aspiration. The observation of clear epithelioid cells can understandably lead to the wrong diagnosis of clear-cell carcinoma of the pancreas and thus lead to duodenocephalopancreasectomy.
A primary angiomyolipoma of the pancreas has also been described. It was a cephalopancreatic nodule in a non-TSC woman [51].
Liver
Apart from kidney, liver is the most likely organ involved by angiomyolipoma (Fig. 13), either classic or epithelioid [44, 114, 118]. Frequently, hepatic AML shows a prominent component of large epithelioid cells [114]. There has also been described a case of CCST of the common bile duct [98].
Fig. 13Hepatic angiomyolipoma: solid component made of large epithelioid cells; H&E ×10
Other sites
Lymphangioleiomyomatosis has also been reported in extrapulmonary sites including mediastinal and retroperitoneal lymph nodes, soft tissue of the mesentery and the renal sinus, as previously described. Usually, extrapulmonary LAM presents as a localized well-circumscribed mass called “lymphangiomyoma” [78].
Apart from kidney and liver, classic or even epithelioid angiomyolipoma can occur in different visceral and somatic sites as gastrointestinal tract, pelvis [14, 34, 47], nasal cavity [7], soft tissues [49] retroperitoneum [63], bone [53] and orbit [54].
Clear-cell “sugar” tumor has been described in a variety of extrapulmonary sites, apart from uterus and pancreas: skin [26], soft tissues [40], breast [45], skull base [66], gastrointestinal tract [8, 111], inter-atrial cardiac septum [111] and oral mucosa [61].
Clear-cell myomelanocytic tumour of the falciform ligament/ligamentum teres (CCMMT) is a PEComa showing predominantly spindled cell morphology.
It has been first described in 2000 by Folpe et al. [36] who presented seven cases of PEComa arising in the abdomen. All patients but one were women ranging from 3 to 21years of age. These neoplasms were composed of spindle cells arranged in fascicles and nests, positive for HMB45 and, in three cases, also for Melan-A, microophtalmia transcription factor (Mitf) and actin. Also in these cases, premelanosomes were ultrastructurally detected. At follow-up, in one patient presumed lung metastasis developed.
CCMMT has also been described in thigh [38] and in the skin [79].
PEComa: the future
PEComas are a group of ubiquitous neoplasms sharing morphological, immunohistochemical, ultrastructural and genetic distinctive features.
There are some open questions about PEComas: the histogenesis and the normal/physiological counterpart of PEC, the definition of epithelioid AML and the identification of the histological criteria of malignancy.
The histogenesis and the normal/physiological counterpart of PEC are unknown, but some hypotheses have been proposed. One hypothesis is that PEC derives from undifferentiated cells of the neural crest that can express dual smooth muscle and melanocytic phenotype; a second hypothesis is that PEC has a myoblastic, smooth muscle origin with a molecular alteration that brings to expression of melanogenesis and melanocytic markers [107]; a third hypothesis is that PEC has a pericytic origin. As regards PEComa’s histogenesis, the involvement of TSC pathway in these neoplasms can suggest some possibilities: it has been previously proposed that B-raf activity in cells lacking TSC2 may play a role in cell differentiation [56]. Moreover, TSC pathway regulates negatively Wnt/beta-catenin pathway [71] and beta-catenin regulates transcription of genes involved in cell proliferation and differentiation. Nevertheless, more cases should be analysed to better understand PEC origin and PEComa’s histogenesis.
The second issue regarding PEComas is which cases should be classified as epithelioid AML particularly in the kidney and liver, where AML occurs with higher frequency. We defined epithelioid AML as a neoplasm composed of purely epithelioid cells with melanogenesis markers immunoreactivity arranged in sheets, without adipocytes and abnormal blood vessels.
However, in otherwise classic AML, areas of epithelioid cells can be observed, raising the question how much they should be represented to call a tumor “epithelioid angiomyolipoma”. The collection of such cases and a consensus meeting could be useful tools to answer to this problem.
Malignant PEComa can be a very aggressive disease leading to multiple metastases and death as expected with a high-grade sarcoma [3, 14, 16, 32, 39, 52, 66, 67, 70, 85, 96, 99, 109, 121, 124]. A few malignant PEComas metastasised after several years (7–9 years) [27, 73, 89, 100] (Table 1).
Table 1Reported cases of malignant PEComas in literatureReferenceDiagnosisSiteSex/ageOutcomeCommentsSale and Kulander [100]CCSTLungn.a./n.a.AWD at 10 yearsHepatic metastases at 10 yearsFerry et al. [32]Epithelioid AMLKidneyF/49DOD at 5.5 months Al-Saleem et al. [3]Epithelioid AMLKidneyF/21DOD at 3 months Pea et al. [96]Epithelioid AMLKidneyF/24DOD at 1 yearPelvic and hepatic metastasesPea et al. [96]Epithelioid AMLKidneyM/29DOD at 18 monthsPulmonary and hepatic metastasesChristiano et al. [21]Epithelioid AMLKidneyM/42AWD at 15 months L’Hostis et al. [67]Epithelioid AMLKidneyF/71DOD at 2 years Folpe et al. [36]PEComaLigamentum teres falciform ligamentM/29DOC at 1 yearRadiographically suspected lung metastasis at 3 monthsMartignoni et al. [73]Epithelioid AMLKidneyF/50AWD at 10 yearsLocal recurrence at 7 years; pulmonary, pelvic and abdominal metastases at 10 yearsBonetti et al. [14]PEComaUterusF/41ANED at 6 monthsOvarian mass at presentationBonetti et al. [14]PEComaUterusF/19AWD at 18 monthsAggressive local recurrence at 1 month; lung and bone metastases at 11 monthsBonetti et al. [14]PEComaTerminal ileum and cecumF/28DOD at 28 monthsHepatic metastasisCibas et al. [22]Epithelioid AMLKidneyF/49AWD at 6 months Saito et al. [99]CCSTKidneyF/23DOD at 1 yearRetroperitoneal recurrence at 3 monthsYamamoto et al. [122]Epithelioid AMLKidneyM/47DODDOD with spinal, lung and lymph node metastases (autopsy case)Dimmler et al. [27]PEComaUterusF/61AWD at 7 yearsLung metastasesGreene et al. [46]PEComaUterus and pelvic side wallF/79DOD at several monthsPelvic and mesenteric recurrence at 2 yearsLau et al. [63]Epithelioid AMLRetroperitoneumM/29ANED at 18 yearsLiver metastases at 9 years; thymus and lung metastases at 17 yearsLeclerc et al. [64]Epithelioid AMLKidneyF/36ANEDLymph node metastasesLin et al. [69]Epithelioid AMLKidneyn.a./58AWDLiver and lymph node metastasesOng et al. [82]Epithelioid AMLKidneyF/74AWDAdrenal metastasesPan et al. [85]PEComaProstateM/46DOD at 4 yearsSubmitted to adjuvant chemotherapy; lung metastases at 3 yearsTakahashi et al. [109]Epithelioid AMLKidneyF/40DOD at 18 monthsSubmitted to adjuvant chemotherapy; pulmonary metastasesTakahashi et al. [109]Epithelioid AMLKidneyM/44DOD at 60 monthsSplenic and retroperitoneal metastases at 3 years; hepatic metastases at 60 monthsYanai et al. [123]PEComaJejunumF/32AWD at 25 monthsPelvic wall recurrence/local metastases at 13 months; ovarian metastases at 25 monthsDarai et al. [24]Epithelioid AMLUrachF/n.a.AWDLymph node metastasesHarris et al. [49]PEComaSoft tissue near kneeM/87AWD at 40 monthsInguinal lymph node and lung metastases at 13 monthsLehman [66]PEComaSkull baseF/49DOD at 3 monthsParaspinal and lung metastasis at 6 weeksBosincu et al. [16]PEComaUterusF/59DOD at 1 yearPelvic recurrence at 6 monthsEvert et al. [30]PEComaRectovaginal spaceF/56AWDPulmonary metastases at presentationFolpe et al. [39]PEComaNeckF/77ANED at 6 monthsRe-excision and submitted to adjuvant radiotherapyFolpe et al. [39]PEComaForearmM/71ANED at 10 monthsRe-excision and submitted to adjuvant radiotherapyFolpe et al. [39]PEComaBroad ligamentF/16ANED at 18 monthsRe-excisedFolpe et al. [39]PEComaFalciform ligamentF/15ANED at 35 monthsRe-excisedFolpe et al. [39]PEComaUterusF/56AWD at 11 yearsSubmitted to adjuvant radio and chemotherapy; lung and bone metastasesFolpe et al. [39]PEComaPelvic soft tissueF/72AWD at 15 monthsLocal recurrenceFolpe et al. [39]PEComaOmentumM/40AWD at 24 monthsExtensive intra-abdominal recurrence/local metastasesFolpe et al. [39]PEComaUterusF/59AWD at 30 monthsSubmitted to adjuvant chemotherapy; liver and lung metastases at 30 monthsFolpe et al. [39]PEComaMesenteryF/46DOD at 27 monthsSubmitted to adjuvant chemotherapy; extensive intra-abdominal recurrence and liver metastases at 22 monthsFolpe et al. [39]PEComaUterusF/36DOD at 39 monthsSubmitted to adjuvant chemotherapy; lung metastases at 12 months and liver metastases at 36 monthsSvec and Velenska [108]Epithelioid AMLKidneyF/47ANED at 5 monthsRecurrence at 3 monthsYu et al. [124]Epithelioid AMLKidneyF/12DOD at 9 monthsLymph node metastases; submitted to adjuvant chemotherapyAgaimy and Wünsch 2006 [2]PEComaIleumF/63AWD at 14 monthsAbdomino-pelvic recurrenceKim et al. [60]CCMTBroad ligamentF/12AWD at 1 yearIliac fossa recurrenceMai and Belanger [70]PEComaThighM/56DOD at 1 yearPulmonary and brain metastasesMai and Belanger [70]PEComaThighF/60DOD at 1 yearPulmonary metastasesMai and Belanger [70]PEComaGroinM/46DOD at 2 yearsLymph node metastasesParfitt et al. [89]PEComaLiverF/60AWD at 10 yearsHepatic recurrence and pulmonary, pancreatic and muscular metastases at 9 years; bladder metastases at 10 yearsParfitt et al. [91]PEComaLung/Adrenal glandF/53AWDBrain metastases at several mothsYamamoto et al. [121]PEComaColonF/43DOD at 38 monthsPeritoneal dissemination at 20 monthsGupta et al. [48]Epithelioid AMLRetroperitoneumF/80AWD at 1 yearHepatic and rib metastasesHuang et al. [52]Epithelioid AMLKidneyF/78DOD at 5 months Park et al. [92]Epithelioid AMLKidneyM/69AWD at 8 monthsHepatic and peritoneal metastasesPark et al. [92]Epithelioid AMLKidneyF/46AWD at 12 monthsHepatic and lymph node metastasesWeinreb et al. [120]PEComaRetroperitoneumF/49AWDBrain and lung metastasesANED Alive, not evidence of disease; AWD alive with disease; DOC dead of other causes; DOD dead of disease; n.a. not available
Recently, Folpe et al. [39] reported 26 cases of PEComas of soft tissue and gynaecologic origin proposing criteria for the classification of these tumors as “benign”, “of uncertain malignant potential” and “malignant”. In this study, they observed a significant association between tumor size >5cm, infiltrative growth pattern, high nuclear grade, necrosis and mitotic activity >1/50 HPF and subsequent aggressive clinical behaviour of PEComas.
We think that this approach is the best available at the moment. While more cases with long follow-up are needed to verify the effectiveness of this prognostic classification, we believe that all cases of PEComa should be classified according to the criteria proposed by Folpe et al.
Another future challenge regarding PEComas is their management.
Surgery seems to be the only approach for aggressive cases, as chemio- and radiotherapy has not shown significant results. However, this derives from anecdotical cases as no therapeutic trial has so far been implemented. There are obvious difficulties to perform a therapeutic trial mainly due to the rarity of the disease. An international cooperative study is needed to address this problem.
A very different problem is posed by lymphangioleyomyomatosis. In fact, lymphangioleyomyomatosis is composed of a population of cells with no atypia. Mitotic activity is virtually absent or extremely low. In spite of this, the disease usually progresses inexorably towards lung-function impairment.
Recently, Kenerson et al. [59] demonstrated TSC1/2 inactivation and m-TOR hyperactivation in non-TSC AMLs and in extrarenal PEComas using immunohistochemistry and Western blot analysis. In particular, m-TOR hyperactivation can be studied in such lesions using immunohistochemical detection of p70S6K. As we have previously mentioned, p70S6K is a protein kinase activated by m-TOR. Immunohistochemical reaction for p70S6K can stain both p70S6K, which gives a cytoplasmic signal (Fig. 14), and a second isoform of kinase, p85S6K, which is also activated by m-TOR and which gives a nuclear signal.
Fig. 14Hepatic angiomyolipoma: cytoplasmic signal in immunohistochemical reaction for p70S6K; p70S6K ×10
Rapamycin is a specific inhibitor of m-TOR [23] which is approved by the US Food and Drug Administration for immunosuppression therapy after renal transplant [117], and it has recently been approved also for therapy of renal cell carcinomas and acute myeloid leukemia [88, 97]. Preclinical studies in animal models of TSC have shown significant in vivo response to rapamycin [58, 65].
As regards LAM, an innovative therapeutic trial is under way. Rapamycin seems to block signaling through the Akt cell growth and survival pathway (for up-to-date information, see http://www.thelamfoundation.org).
A similar trial has already shown a positive effect on renal AML, and we can hope to see the same effect on LAM.
If this will be the case, this could provide the rationale for the use of the same drug in other lesions composed of PECs.

pecoma
pec
tsc
mtor
angiomyolipoma
rapamycin
lymphangioleiomyomatosis
sugar tumor


Identification of InuR, a new Zn(II)2Cys6 transcriptional activator involved in the regulation of inulinolytic genes in Aspergillus niger

The expression of inulinolytic genes in Aspergillus niger is co-regulated and induced by inulin and sucrose. We have identified a positive acting transcription factor InuR, which is required for the induced expression of inulinolytic genes. InuR is a member of the fungal specific class of transcription factors of the Zn(II)2Cys6 type. Involvement of InuR in inulin and sucrose metabolism was suspected because of the clustering of inuR gene with sucB, which encodes an intracellular invertase with transfructosylation activity and a putative sugar transporter encoding gene (An15g00310). Deletion of the inuR gene resulted in a strain displaying a severe reduction in growth on inulin and sucrose medium. Northern analysis revealed that expression of inulinolytic and sucrolytic genes, e.g., inuE, inuA, sucA, as well as the putative sugar transporter gene (An15g00310) is dependent on InuR. Genome-wide expression analysis revealed, three additional putative sugar transporters encoding genes (An15g04060, An15g03940 and An17g01710), which were strongly induced by sucrose in an InuR dependent way. In silico analysis of the promoter sequences of strongly InuR regulated genes suggests that InuR might bind as dimer to two CGG triplets, which are separated by eight nucleotides.
Introduction
Some plants store part of their carbon as sucrose and as polymers of fructose (fructans). Plant fructans, often referred to as inulins, have predominantly β-2,1 linkages between the fructose moieties and terminate with a sucrosyl residue. Inulin is present in a range of plant species. In some species such as chicory, garlic, Jerusalem artichoke and Dahlia tubers about 15–20% of the plant fresh weight consists of inulin (Gupta and Kaur 1997; Kaur and Gupta 2002). Apart from the use of inulin for the production of fructose rich syrups, inulin and fructo-oligosaccharides (FOS) have attracted considerable research attention because of the large number of health benefits obtained from inulin and FOS in the diet of humans (see for a review Kaur and Gupta 2002).
Aspergillus niger is a saprophytic fungus, mainly present in the soil, feeding preferably on organic matter such as plant cell wall polysaccharides (cellulose, hemi-cellulose and pectin), and on plant storage polysaccharides (starch and inulin). A. niger is able to produce various inulin degrading enzymes including an exo-inulinase, an endo-inulinase and an invertase (Moriyama et al. 2003; Ohta et al. 1998; Akimoto et al. 1999; Berges et al. 1993; Boddy et al. 1993; L’Hocine et al. 2000). The inulinolytic enzymes are simultaneously induced in the presence of inulin or sucrose, indicating that the expression of the genes encoding these enzymes is co-regulated and controlled by a common transcription factor (Yuan et al. 2006).
Transcriptional activation of catabolic enzyme networks is often accomplished through transcription factors of the Zn(II)2Cys6 type. This type of transcription factor is unique to fungi. Gal4 is the transcriptional activator for the expression of genes involved in galactose utilization, and is the best characterized Zn(II)2Cys6 transcription factor. In general, Zn(II)2Cys6 transcription factors possess a well conserved N-terminal localized DNA binding motif (CX2CX6CX5–16CX2CX6–8), followed by a less well defined conserved domain known as a fungal specific transcription factor domain (Pfam04082). The C-terminal part of the Zn(II)2Cys6 transcription factor normally contains the activation domain. In the genome sequence of Saccharomyces cerevisiae, 54 putative Zn(II)2Cys6 transcription factors have been identified (Akache et al. 2001). They are involved in the transcriptional control of a wide variety of cellular processes (Todd and Andrianopoulos 1997; Akache et al. 2001) including genes involved in amino acid metabolism (ArgRp, Crabeel et al. 1995; Leu3p, Kohlhaw 2003; Lys14p, El Alami et al. 2000), sugar metabolism (Mal63p, Ni and Needleman 1990), pyrimidine biosynthesis (Ppr1p, Flynn and Reece 1999). In the genome of filamentous fungi even more Zn(II)2Cys6 transcription factors are present. In the recently released genome sequence of A. niger CBS 513.88, 296 ORFs were identified that contain the Zn(II)2Cys6 motif and similar numbers are predicted from the genomes of A. fumigatus and A. nidulans (see for comparison Pel et al. 2007). The role of only a few of the Zn(II)2Cys6 transcription factors in filamentous fungi have been studied in detail. Filamentous fungal Zn(II)2Cys6 proteins are involved in the regulation of various processes which include secondary metabolite production (e.g. AflR, Woloshuk et al. 1994), pigmentation (e.g. Cmr1, Tsuji et al. 2000), nitrogen metabolism (NirA, Burger et al. 1991), proline utilization (PrnA, Cazelle et al. 1998; Leucine biosynthesis (Leu3p, Kirkpatrick and Schimmel 1995), alcohol utilization (AlcR, Kulmburg et al. 1992) and sexual development (RosA, Vienken et al. 2005). Well studied examples of fungal Zn(II)2Cys6 transcription factors required for polysaccharide catabolism include AmyR, a transcriptional activator of starch degrading enzymes (Petersen et al. 1999; Gomi et al. 2000; Tani et al. 2001a), and XlnR, a transcriptional activator for xylanolytic enzymes (van Peij et al. 1998a, 1998b; Gielkens et al. 1999; Hasper et al 2004).
It is well established that the genes encoding many proteins required for secondary metabolite production are clustered in the genome (Woloshuk et al. 1994; Walton 2000). In addition to several biosynthetic enzymes that synthesize the secondary metabolites, such clusters also contain a Zn(II)2Cys6 transcription factor which is required for the transcriptional activation of the genes within the cluster. Gene clustering is not limited to secondary metabolite pathways, but also found for the proline utilization genes (PrnA, Gomez et al. 2002) and for some of the amylolytic genes under the control of the regulator AmyR. Adjacent to the gene for the AmyR transcription factor are genes encoding alpha-glucosidase (AgdA) and an alpha-amylase (AmyA) (Gomi et al. 2000). Also in A. parasiticus, a sugar utilization gene cluster has been identified (Yu et al. 2000). However, it is clear that clustering of genes with related functions is not a general rule, since the genes adjacent to the XlnR transcription factor do not seem to be involved in xylan degradation.
In a previous study, we showed that the expression of the genes encoding inulin degrading enzymes is co-regulated and specifically induced in the presence of inulin and sucrose (Yuan et al. 2006). The availability of the genome sequence of A. niger allowed the rapid identification of Zn(II)2Cys6 transcription factors adjacent to or in close proximity to the genes encoding (putative) enzymes involved in inulin catabolism. We have identified a Zn(II)-cluster transcription factor (InuR) which is adjacent to a gene encoding a sugar transporter and a recently identified intracellular invertase (SucB) with transfructosylation activity (Goosen et al. 2007).
In this paper, we show that deletion of the inuR gene results in strongly impaired growth on inulin and sucrose, and also abolishes the induction of the genes encoding the extracellular enzymes involved in inulin and sucrose degradation. The gene encoding the putative sugar transporter located next to inuR is co-regulated with the genes encoding the inulinolytic enzymes. These results show that InuR acts as a positive acting transcriptional activator for the induced expression of genes involved in the breakdown of inulin and sucrose and the uptake of inulin breakdown products.
Materials and methods
Strains, culture conditions and transformation
Aspergillus niger strain N402 used in this study was derived from the wild-type strain A. niger van Tieghem (CBS 120.49, ATCC 9029) (Bos et al. 1988). The A. niger strain used by DSM to sequence the genome is NRRL 3122. Strain AB4.1 is a pyrG negative derivative of N402 (van Hartingsveldt et al. 1987) and was used to construct disruption strains. A. niger strains were grown in Aspergillus minimal medium (MM) (Bennet and Lasure 1991), or Aspergillus complete medium (CM) consisting of MM medium with the addition of 0.5% (w/v) yeast extract and 0.1% (w/v) casamino acids. Growth medium was supplemented with 10 mM uridine (Serva) when required. For shake flask cultures, A. niger strains were grown in MM supplemented with 1% (w/v) carbon source and 0.1% (w/v) casamino acids. The conidiospores were inoculated at a concentration of 2 × 106 spores per ml. Glucose and sucrose (BDH chemicals), maltodextrin (Avebe), starch (Windmill Starch, Avebe), xylose, fructose and maltose (Sigma-Aldrich), raffinose (Sigma chemicals) and inulin (Sensus Frutafit, Cosun) were used as carbon sources.
For transfer experiments, A. niger strains were pre-grown in MM supplemented with 2% (w/v) xylose and 0.1% (w/v) casamino acids for 18 h at 30°C on a rotary shaker at 300 rpm. Then mycelium was harvested by suction over a nylon membrane and washed with MM without carbon source. Aliquots of 1.6 g wet weight of mycelium were transferred to 300-ml Erlenmeyer flasks containing MM supplemented with 1% (w/v) carbon source and incubated at 30°C for the time indicated in the text. The mycelium was harvested over Myracloth (Calbiochem) and frozen in liquid nitrogen, followed by storage at −80°C prior to the isolation of total RNA. Conidiospores were obtained by harvesting spores from CM plates after 4–6 days of growth at 30°C, using a 0.9% NaCl solution. Transformation of A. niger AB4.1 was done as described by Punt and van den Hondel (1992) using lysing enzymes (L1412, Sigma) for protoplastion. The bacterial strain used for transformation and amplification of recombinant DNA was Escherichia coli XL1-Blue (Stratagene). Transformation of XL1-Blue was performed according to the heat shock protocol as described by Inoue et al. (1990).
Construction of the inuR::pyrG deletion strain
The A. nigerinuR gene was deleted by the replacement of the complete open reading frame of the inuR gene with the Aspergillus oryzaepyrG gene. The plasmid used to disrupt inuR was constructed as follows. The DNA fragments flanking the inuR ORF were amplified by PCR using N402 genomic DNA as template. Fragments with a length of 0.8 kb of 5′ flanking DNA and 1.0 kb of 3′ flanking DNA were amplified using primers inuRP1 and inuRP2, inuRP3 and inuRP4 (Supplementary Table 1), respectively. Each primer was adapted with restriction sites as indicated for further cloning. The amplified PCR fragments were digested with NotI and XhoI or XhoI and KpnI, respectively, and cloned into pBlue-Script II to obtain plasmid pInuRF5 and pInuRF3. Subsequently, the 0.8 kb NotI-XhoI fragment containing 5′ flanking of inuR from pInuRF5, was ligated into NotI/XhoI digested pInuRF3 to give pInuRF53. The A. oryzaepyrG gene was isolated as a 2.8 kb SalI–BamHI fragment, obtained from plasmid pAO4-13 (de Ruiter-Jacobs et al. 1989) and ligated into XhoI/BamHI digested pInuRF53 to obtain pΔinuR (pXY3.1). Before transformation, the plasmid pΔinuR was linearized with NotI and transformed into A. nigerpyrG- strain AB4.1. Uridine prototrophic transformants were selected by incubating protoplasts on agar plates containing MM without uridine. Transformants were screened by PCR using primers PAO9 and inuRP5 or PAO10 and inuRP6 (Supplementary Table 1). Only transformants with a targeted deletion of the inuR gene should result in the amplification of a 1.2 or 1.4 kb PCR fragment, respectively. PCR positive transformants were verified by Southern blot analysis as described by Sambrook et al. (1989).
Chromosomal DNA from positive PCR transformants was isolated as described by Kolar et al. (1988). Ten μl of genomic DNA was digested for 3 h with 10 U of XhoI or EcoRI, respectively. The 1.0 kb of the 3′-flanking DNA fragment of inuR was used as probe for the detection of inuR disruptants. A probe was generated by digestion of the pInuRF3 plasmid containing the inuR 3′-flanking fragment with XhoI and BamHI. Fragments were purified from gel and [α-32P]dCTP labeled probes were synthesized using Rediprime II DNA labeling System (Amersham Pharmacia Biotech) according to the instructions of the manufacturer.
Complementation of inuR mutant
The A. niger inuR gene was amplified by PCR using primers inuRP7 and inuRP8 (Supplementary Table 1) and N402 genomic DNA as a template. The primers corresponded to 1 kb upstream and 0.5 kb downstream of the A. niger inuR gene, respectively. PCR was performed using Phusion™ High-Fidelity DNA Polymerase (Finnzymes) according to the manufacturer’s instruction. The amplified 4.1 kb PCR fragment was cloned into pGEMT-easy vector (Promega) to obtain the inuR complementation plasmid pXY5.1. pXY5.1 was then co-transformed with pAN7.1 harboring hygromycin gene as selection marker (Punt et al. 1987) into A. niger ΔinuR strain XY3.1 (see in the text). Hygromycin resistant transformants were selected by incubating protoplasts on agar plates containing MM with 200 mg/ml of hygromycin. Southern blot analysis was performed on selected transformants as described by Sambrook et al. (1989).
Construction of the ΔinuR ΔamyR double deletion strain
To make a pyrG-strain of the ΔinuR strain (XY3.1) this strain was incubated on MM plates containing 1 mg/ml of 5′FOA and 10 mM uracil. 5′FOA resistant, uracil requiring mutants were transformed with pAB4.1 harboring the A. nigerpyrG or with pPyrE harboring the A. niger pyrE gene on a 4.3 kb SstII subclone, respectively. One of the selected 5′FOA resistant mutants, XY4.1, was complemented with the pyrG gene, indicating that the uracil auxotrophy was caused by a mutation in the pyrG gene. The strain XY4.1 was then used to construct a ΔinuR ΔamyR double disruption strain by transforming plasmid pΔamyR which also contains the pyrG selection marker (Yuan et al., submitted). Uridine prototrophic transformants were selected by incubating protoplasts on agar plates containing MM without uridine. Transformants that showed normal growth on glucose but not on starch were selected. One possible ΔinuRΔamyR double mutant (XY5.1) was verified by Southern analysis and used for further analysis.
Northern blot analysis
Total RNA isolation, Northern analysis and synthesis of DNA probes was performed as described in Yuan et al. (2006). The primers used to generate probe An15g00310 are shown in Supplementary Table 1.
Microtiter plate growth assay
Growth of A. niger strains were determined using a HTS7000 BioAssay Reader (Perkin Elmer Life Sciences). Spores (1 × 104) were inoculated in each well of a 96-well microtiter plate (Nalge Nunc International, USA) and incubated at 32°C for 56 h. Each well contained 200 μl of MM containing 1% (w/v) of one of the various carbon sources each supplemented with 0.01% (w/v) casamino acids to stimulate spore germination. Six replicates of each condition were made. Growth was monitored by measuring turbidity (OD595) at 2-h intervals.
Microarray experiments and data analysis
RNA extracted from the A. niger ΔinuR strain and wild-type strain (N402) grown on different carbon sources were used for microarray experiments using custom made “dsmM_ANIGERa_coll” Affymetrix GeneChip® microarrays kindly provided by DSM Food Specialties (Delft, The Netherlands). For microarray experiments, mycelia were isolated from grown cultures by transfer experiment (see above) and each growth condition was performed in duplicate as independent biological experiments.
Total RNA was isolated from mycelia using TRIzol reagent (Invitrogen) and RNA quality was verified by analyzing aliquots with glyoxal/DMSO gel electrophoresis and Agilent Bioanalyzer “Lab-on-chip” system (Agilent Technologies, USA). Processing, labeling and hybridization of cRNA to A. niger Affymetrix GeneChips were performed according to the corresponding Affymetrix protocols for “Eukaryotic Target Preparation” and “Eukaryotic Target Hybridization”. For probe array washing and staining, the protocol “Antibody Amplification for Eukaryotic Targets” was followed. Hybridized probe array slides were scanned with Agilent technologies G2500A Gene Array Scanner at 3 μm resolution and a wavelength of 570 nm. Affymetrix Microarray Suite software MAS5.0 was used to calculate the signal and P values and to set the algorithm’s absolute call flag, which indicates the reliability of the data points according to P (present), M (marginal) and A (absent). The data on each chip were globally scaled to an arbitrary target gene intensity of 500.
The pre-scaled chip data from each hybridization experiment were normalized using Genespring 7.0 software (Silicon Genetics). Normalization with default parameters in Genespring software (Per Chip: Normalize to 50th percentile, Per Gene: Normalize to median) was used. For this study, we focused on sucrose induced genes. Therefore, pre-filtering of data was performed to select for genes whose detection calls are present in both sucrose duplicate samples of wild-type strain N402. The selected data set was further performed for one-way ANOVA analysis. Fold changes in expression levels between two different conditions were then computed for genes with P < 0.05 based on One-way ANOVA analysis and changes by more than twofold were considered significant and are reported here. Microarray data were deposited into ArrayExpress with an accession number 19095 at http://www.ebi.ac.uk/at-miamexpress.
In silico analysis of regulatory elements of co-regulated genes
The 1.0 kb promoter sequences of the seven sucrose induced InuR dependant genes: An12g08280 (InuE), An11g03200 (InuA), An08g11070 (SucA), An15g04060, An15g00310, An15g03940, and An17g01710 were obtained from the CBS513.88 strain. The 1.0 kb promoter regions were analyzed on both strands using DNA Analyzer module of Phylosopher 6.5.1 (Genedata A.G., Basel, Switzerland) using the Gibbs Sampling Strategy for Multiple Alignment (Lawrence et al. 1993).
Results
Identification of potential transcriptional regulators involved in inulin catabolism
A. niger is able to produce various inulinolytic enzymes involved in the modification or degradation of inulin and sucrose. The transcriptional regulation of the genes encoding the inulinolytic enzymes of A. niger, including an exo-inulinase (InuE; An12g08280), an endo-inulinase (InuA; An11g03200), and an invertase (SucA; An08g11070), has been studied previously (Moriyama et al. 2003; Yuan et al. 2006). In addition to the extracellur enzymes, two additional potential intracellular inulinolytic enzymes (SucB; An15g00320 and SucC; An06g02420) were predicted from the A. niger genome sequence (Yuan et al. 2006). These proteins contain all the conserved domains of the GH32 family to which inulinolytic enzymes belong. Finally, a potential ORF was found in the genome which showed homology to the group of exo-inulinases. Because the predicted ORF lacked several of the conserved domains and contained several frame shift mutations, this gene (inuQ, An11g03210) was considered to be a pseudogene (Yuan et al. 2006).
The expression of the genes encoding the extracellular inulinolytic enzymes is co-regulated and induced on inulin and sucrose, suggesting that these genes are under control of a single transcription factor (Yuan et al. 2006). Some transcriptional activators involved in sugar catabolism are clustered in the genome with their target genes (Gomi et al. 2000; Yu et al. 2000). To identify possible candidate transcription factors involved in the regulation of inulinolytic genes, we searched the A. niger genome sequence for transcription factors located close to the genes encoding the inulinolytic enzymes. We found that inuA (An11g03200) and inuQ (An11g03210) are located next to each other and that also a Zn(II)2Cys6 transcription factor encoding gene (An11g03220) was located adjacent to inuQ. The orientation of the three ORFs is in the same direction indicating that they do not share a common promoter region (Fig. 1).
Fig. 1Schematic representation of the clustering of putative transcription factors with inulinolytic genes in the genome of A. niger. An11g03220 and An08g11040 and inuR encode putative transcription factors. The genes related to inulin degradation include: inuA (endo-inulinase), inuQ (pseudogene), sucA (invertase), sucB (homologous to sucA) and An15g00310 (putative sugar transporter). An08g00160 encodes a hypothetical protein without known function. The arrows indicate the transcriptional orientations of the genes
In the proximity of sucA (An08g11070), a second transcription factor encoding gene was found. This transcription factor contains a Cys2His2 zinc finger DNA binding motif. Between this gene, An08g11040 and sucA an additional ORF (An08g11060) is present. This 273 amino-acid protein encodes a hypothetical protein without any conserved motifs. A BlastP search revealed that this protein has no close orthologs in other fungal genomes. The transcription factor, encoded by An08g11040, shows strong sequence similarity to the S. cerevisiae Zpr1p (e-value 1e-100), an essential transcription factor that contributes to normal cell proliferation (Gangwani et al. 1998).
Clustered with sucB (An15g00320) a third putative transcription factor encoding gene (An15g00300) was found which also belonged to the Zn(II)2Cys6 transcription factor family. Between sucB and An15g00300, an additional ORF (An15g00310) is located (Fig. 1). The protein encoded by this gene has all the characteristics of a sugar transporter protein. In the direct proximity of the two remaining genes encoding inulinolytic enzymes (InuE and SucC) no potential transcription factors were found. The three transcription factors identified via clustering were considered to be good candidates to be involved in the transcriptional regulation of the inulinolytic system of A. niger. In order to study their possible role in inulin utilization, deletion strains of these transcription factors were constructed. Deletion of An11g03220 did not have any measurable effect on growth on inulin or sucrose in comparison with the wild-type strain, which indicates that this gene is not required for inulin utilization. Growth of the deletion strain on xylose, glucose, fructose, starch and maltose was also identical to the growth of the wild-type strain (data not shown). A deletion strain of An08g11040 was never obtained although over 500 transformants were screened. A possible explanation might be that An08g11040 is an essential gene. As indicated above, An08g11040 is most homologous to the essential Zpr1p transcription factor of S. cerevisiae. Since the homology with Zpr1p does not indicate involvement of this transcription factor in inulin utilization, no further attempts were made to obtain the deletion strain. As shown in detail below, deletion of An15g00300 resulted in a strain which showed a severe growth defect on inulin and on sucrose. For the remainder of the paper, we will refer to An15g00300 as inuR.
InuR homologs were also identified in the genomes of other Aspergilli. The alignment of the InuR proteins is given in Fig. 2. The strong conservation of the transcription factor among Aspergilli, suggest that the regulation of inulinolytic gene expression is mediated in the various Aspergilli by the InuR proteins present in the different fungi.
Fig. 2Alignment of InuRp and its orthologs in Aspergilli. The amino acid sequences of the Zn2Cys6 binuclear DNA-binding motif (Pfam00172) and the Fungal specific transcription factor domain (Pfam04082) are underlined. The conserved six cystein residues are indicated by stars. AoInuRp, AgInuRp, AfInuRp and AdInuRp are InuR protein orthologs from A. oryzae, A. niger, A. fumigatus and A. nidulans, respectively
InuR encodes a Zn(II)2Cys6 transcription factor that is required for growth on inulin
Based on the analysis of the CBS513.88 genomic sequence, the predicted open reading frame of the inuR encoding gene is 2,466 bp long and interrupted by 4 introns with sizes of 153, 80, 58 and 48 bp, respectively. The predicted InuR protein sequence is comprised of 709 amino acid residues which would result in a protein with a calculated molecular mass of 78.3 kDa. Analysis of the predicted InuR protein indicated that InuR contains two conserved domains. One is the Zn(II)2Cys6 (CX2CX6CX5CX2CX6C) binuclear cluster which represents the DNA-binding domain at the NH2-terminal end (residues 35–61). The domain is very well conserved (Pfam00172) and binds two Zn atoms which coordinate folding of the domain. A BlastP search revealed orthologous transcription factors in the genome of A. nidulans, A. oryzae and A. fumigatus (see above, Fig. 2). The most similar transcription factors to InuR which have been functionally characterized, apart from the proposed orthologs in A. oryzae, A. fumigatus and A. nidulans, are the AmyR transcription factors from the different Aspergillus species. Subsequent phylogenetic analysis indicated that the subgroup of InuR transcription factors is most closely related to the group of AmyR transcription factors (data not shown). Besides the Zn(II)2Cys6 motif, a second conserved domain, known as the middle homology region (MHR) or Pfam04082 domain, is present in Zn(II)2Cys6 transcription factors. The region is thought to assist the Zn(II)2Cys6 cluster in DNA target discrimination (Schjerling and Holmberg 1996). In the A. niger InuR protein, the MHR/Pfam04082 domain compromises aa 353–425, and the domain is also present in the InuR proteins of the other Aspergilli.
Construction and growth properties of the inuR disruptant in A. niger N402
An inuR disruptant mutant was constructed as described in the “Materials and methods”. Putative disruption strains were identified by PCR screening, and further verified by Southern blot analysis. Strain XY3.1 was selected to further characterize the InuR function and we will refer to this strain as the ΔinuR strain.
The effect of the disruption of the inuR gene on the utilization of various carbon sources was analyzed on agar plates. Growth and conidiation of the ΔinuR strain was strongly reduced on plates containing inulin in comparison to the wild-type strain (Fig. 3). As shown, growth of the ΔinuR strain on inulin was not completely abolished. After 7 days of growth, a thin colony with a reduced degree of conidiation was formed. Some effect on growth and conidiation was also observed for the ΔinuR strain growing on plates containing sucrose and to a lesser extent on raffinose. The differences in growth on sucrose and raffinose were most clear after 3 days of growth (Fig. 3). Both the colony size and the degree of conidiation of the ΔinuR strain and the wild-type strain after 7 days of growth were very similar (data not shown). The ΔinuR strain showed a similar growth phenotype to the wild-type on MM plates containing fructose, glucose, maltose, starch, xylose, sorbitol or glycerol as sole carbon source (Fig. 3 and data not shown).
Fig. 3Effects of A. niger strains on the utilization of different carbon sources. The wild-type (N402) and inuR disruptant (ΔinuR), amyR disruptant (ΔamyR) and double mutant (ΔamyRΔinuR) strains were grown on MM plates containing 1% different carbon sources at 30°C for 3 days
Growth of the ΔinuR strain on different carbon sources was also determined by analyzing growth in a microtiter plate well containing MM with various carbon sources. Equal numbers of spores of the wild-type strain (N402) and ΔinuR strain were inoculated and plates were incubated at 32°C. For each condition at least four replicates were made and the average value of the replicates and its standard deviation value were determined as shown in Fig. 4. The ΔinuR showed a clear growth defect on inulin and sucrose. A small, but reproducible growth difference in growth on raffinose on agar plates was also observed in the microtiter plate analysis. Initially, the growth curves of the wild-type and the ΔinuR strain on raffinose were identical, but after 24–32 h growth of the ΔinuR strain seem to be retarded (Fig. 4). The ΔinuR strain grew equally well as the wild-type strain on MM with maltodextrin, maltose, glucose, xylose or fructose as sole carbon source (Fig. 4 and data not shown). In conclusion, the growth analysis of the ΔinuR strain indicates that the Zn(II)2Cys6 transcription factor is required for the efficient utilization of inulin and and to a lesser extent raffinose, and it may therefore act as a positive transcriptional activator of the genes encoding the inulinolytic enzymes. To further substantiate that the defective growth of the ΔinuR strain on inulin was caused by the disruption of the inuR gene, a plasmid containing the inuR gene with 1 kb upstream and 0.5 kb downstream region was constructed and co-transformed into the ΔinuR strain with pAN7.1 (Punt et al. 1987). Hygromycin resistant transformants were analyzed for growth on inulin and subjected to Southern blot analysis. Transformants that showed restored growth on inulin all harbor the inuR gene, while transformants showing defective growth on inulin did not contain the inuR gene (data not shown). The restoration of the wild-type growth on inulin upon retransformation of the ΔinuR strain with a functional copy of the inuR gene indicated that the inability to grow on inulin was indeed a consequence of inuR disruption.
Fig. 4Microtiter plate assay of growth of inuR disruptant on different carbon sources. A. niger wild-type (N402; open circles) strain and inuR disruptant (ΔinuR; solid circles) were inoculated in microtiter plate wells and each well was inoculated with 1 × 104 spores in 200 μl of MM containing 1% carbon source and 0.1% casamino acids. The growth was monitored by measuring culture turbidity (OD595) and read every 2 h. Each condition was performed in six replicates. The standard deviation value is indicated
InuR is required for the induction of inulinolytic genes
To further establish the role of InuR as a positive acting transcriptional regulator for inulinolytic gene expression, Northern blot analysis was performed to determine the mRNA level of the various inulinolytic enzyme encoding genes both in the ΔinuR strain and the WT strain. We previously showed that the expression of the genes encoding the exo-inulinase (inuE), endo-inulinase (inuA), invertase (sucA), was detected only in the presence of inulin and sucrose and not on other carbon sources tested such as xylose, glucose, maltose or starch. Expression of the sucB gene was low on all the carbon sources tested and slightly induced in the presence of inulin and sucrose (Yuan et al. 2006).
In order to clarify whether the expression of these inulinolytic genes was controlled by InuR, both the wild-type strain and the ΔinuR strain were pre-grown for 18 h in MM containing xylose as a carbon source. After the pre-growth, equal amounts of mycelium was transferred to fresh MM containing 1% (w/v) inulin or 1% (w/v) sucrose as a carbon source. At different time points after the transfer RNA was extracted from mycelia and subjected to Northern blot analysis (Fig. 5). The transfer to inulin and sucrose resulted in an induction of the inulinolytic enzymes in the wild-type strain. The induction of genes is much faster on sucrose than on inulin and is explained by a much more rapid degradation and uptake of the carbon source as observed previously (Yuan et al. 2006). In the ΔinuR strain, no induction of expression was found for the inuE and inuA genes after transfer to inulin and a very low level of sucA mRNA was detected after the transfer to inulin. The transfer to sucrose did not result in a detectable induction of any of the three inulinolytic genes in the ΔinuR strain. A slight induction of expression of sucB in the presence of inulin and sucrose seems also dependent on InuR (Fig. 5). The expression of the gene encoding a putative sugar transporter, An15g00310 in relation to inulin and sucrose was also examined. As discussed above, An15g00310 is clustered and located between the inuR and the sucB genes (Fig. 1). Expression of An15g00310 is strongly induced in response to the presence of inulin and sucrose and dependant on the InuR transcription factor (Fig. 5). No or very low expression levels of An15g00310 were detected when grown on maltose, glucose, fructose or xylose or after a transfer to maltose indicating that An15g00310 expression is specific for growth on inulin and sucrose (data not shown). The inuR gene was constantly expressed at a relatively low level on the various carbon sources tested (inulin, sucrose, maltose, fructose or glucose, data not shown), suggesting that the activation of the InuR transcription factor is regulated post-transcriptionally.
Fig. 5Northern blot analysis of inulinolytic genes in wild-type (N402) and the ΔinuR strain from mycelia transferred from xylose (preculture) to inulin or sucrose. Total RNA was isolated from mycelia growing in inulin or sucrose at different time points (2, 4, 8 and 24 h) as indicated after the transfer
Genome wide transcriptional profiling of the InuR regulon in A. niger
The experiments described above suggest that InuR is a transcriptional activator of inulinolytic genes and required for the efficient utilization of inulin and sucrose. In addition to regulating the inulinolytic genes, it is possible that InuR also regulates other genes in the genome, through direct or indirect processes in the pathway of inulin or sucrose utilization. To identify additional genes with a possible role in inulin or sucrose metabolism, we performed genome wide expression analysis using A. niger Affymetrix GeneChip arrays. The A. niger wild-type strain N402 and the ΔinuR strain were pre-grown for 18 h in 2% (w/v) xylose and transferred to fresh medium containing 1% (w/v) sucrose as carbon source and grown for 2 h on sucrose. As a control, the mycelium from the N402 strain was also transferred to fresh 1% (w/v) xylose medium. For each culture condition a duplicate experiment was carried out. RNA was isolated from each of the samples and subjected to microarray analysis (see “Materials and methods”).
To identify genes that were induced by sucrose, we first compared the expression profile of A. niger wild-type strain N402 grown in sucrose to that of A. niger wild-type strain N402 grown in xylose 2 h after transfer. The expression of 97 genes was more than twofold increased by sucrose (One-way ANOVA analysis P < 0.05) (Supplementary Table 2). To identify genes that were induced on sucrose in an InuR dependant way, the expression profile of the A. niger wild-type strain N402 grown in sucrose was compared to that of the ΔinuR strain after the 2 h transfer to sucrose. A total of 40 genes were identified where expression was significantly increased on sucrose in the N402 strain, but not in the ΔinuR strain (One-way ANOVA analysis P < 0.05 and >2-fold change) (Supplementary Table 3.) Finally, by combining these 2 data sets, 26 genes were identified for which expression was induced by sucrose in an InuR dependent manner (Table 1).
Table 1Description of sucrose induced and InuR dependent genes in A. niger at 2 h after transfer from precultureORF No.N402 sucrose N402 xylose amyR sucrose Fold inductionP valueFunctionSucrose versus xyloseN402 vs ΔinuRCarbohydrate transport and metabolism An08g11070103.40 ± 21.54aP 0.11 ± 0.01A 0.26 ± 0.45A930.3391.10.008Invertase  An15g0406036.82 ± 1.36P 0.18 ± 0.03A 0.29 ± 0.05A 204.2126.60.009Putative fructose facilitator An12g08280145.30 ± 27.90P 1.81 ± 0.28P, M 1.97 ± 0.21P, M 80.373.80.008Exo-inulinase An11g0320029.27 ± 0.80P 0.63 ± 0.23A 0.74 ± 0.06A46.239.60.007Endo-inulinase An15g0031020.64 ± 2.95P 1.34 ± 0.17A 1.76 ± 0.97P 15.411.70.018Putative monosaccharide transporter  An15g03940167.30 ± 1.20P 65.74 ± 3.04P 61.68 ± 47.66P 2.52.70.027Putative monosaccharide transporter Amino acid transport and metabolism An04g0134044.31 ± 3.39P 16.25 ± 1.27P 10.16 ± 14.17P 2.74.40.041Putative asparagine synthase  An14g027207.75 ± 0.04P 2.37 ± 0.17p 2.88 ± 1.17P 3.32.70.037Putative neutral amino acid permease Lipid transport and metabolism An08g0367010.76 ± 0.09P 4.45 ± 0.14P 3.91 ± 2.36P, A 2.42.80.015Putative ethanolamine kinase  An16g061902.42 ± 0.14P 1.16 ± 0.07P, A 0.97 ± 0.19A 2.12.50.039Putative glycerophosphoinositol transporter Phosphate transport and metabolism An02g001808.04 ± 0.06P 2.85 ± 0.03P 0.50 ± 0.22A 2.816.20.001Putative phosphate-repressible phosphate permease Nucleotide transport and metabolism An02g0996041.96 ± 4.97P 8.61 ± 0.73P 16.54 ± 7.61P 4.92.50.033Putative phosphoribosylformylglycinamidine synthase Other transport mechanism An17g0171054.97 ± 15.19P 2.83 ± 0.65P 5.71 ± 10.10P, A 19.49.60.049Putative multidrug transporter An08g0233026.99 ± 4.40P 5.73 ± 0.31P 12.12 ± 0.63P 4.72.20.024Putative ABC multidrug transporter An17g0024040.24 ± 3.78P 12.14 ± 0.61P 19.54 ± 8.12P 3.32.10.038Putative ABC transporterTranscription An02g054609.25 ± 1.05P 2.60 ± 0.23A 4.26 ± 0.83M, A 3.62.20.040Putative DNA-directed RNA polymerase An02g126103.81 ± 0.25P 1.55 ± 0.02A 1.21 ± 0.89P, A 2.53.20.043Putative Ran-GTPase-activating proteinProtein synthesis An02g1264048.21 ± 6.42P 9.16 ± 1.17P 18.71 ± 1.32P 5.32.60.041Putative RNA-binding protein  An17g0181555.05 ± 1.42P 15.21 ± 0.37P 24.59 ± 6.90P 3.62.20.004Putative translation initiation factor An01g0604010.17 ± 2.79P 3.21 ± 0.04P 3.88 ± 0.04P 3.22.60.025Putative 60S ribosome biogenesis protein An02g1408023.20 ± 0.69P 9.94 ± 0.25P 10.87 ± 7.87P 2.32.10.010Putative asparaginyl-tRNA synthetaseProtein fate An11g0498036.47 ± 1.45P 9.59 ± 1.06P 12.82 ± 0.53P 3.82.80.010Putative importin unit beta-4Unclassified An01g003409.56 ± 0.97P 2.26 ± 0.42P 3.13 ± 0.32P, A 4.23.10.036Unknown protein An15g0315013.42 ± 0.68P 4.01 ± 0.16P 4.02 ± 0.26P 3.43.30.008Unknown protein An07g026905.27 ± 0.03P 1.69 ± 0.02A 2.22 ± 0.27P 3.12.40.001Unknown protein An01g055003.77 ± 0.06P 1.25 ± 0.03P 1.46 ± 0.35P 3.02.60.005Unknown proteinaThe expression level was based on the geometric mean value of the duplicate samples and the deviation values between the duplicate samples are indicated. P, M or A, representing detection calls for present, marginal or absent respectively. The P-value was based on One-way ANOVA analysis. Genes were divided over different functional groups according to FunCat (Ruepp et al. 2004)
The predicted protein sequences of these 26 genes were assigned to various functional categories (Ruepp et al. 2004; Mewes et al. 1997) and shown in Table 1. Interestingly, genes with the highest fold changes (>10-fold change) almost all fall into the category of carbohydrate transport and metabolism. Among them are the genes encoding the extracellular inulinolytic enzymes, sucA, inuE and inuA, as well as the above mentioned putative sugar transporter gene (An15g00310), supporting the previous Northern analysis. An additional gene that showed a very high fold-change and belongs to the category of carbohydrate transport and metabolism is An15g04060. An15g04060 encodes a putative sugar transporter which is over 200-fold induced by sucrose in a wild-type strain N402 when compared to xylose and the induction was greatly reduced by 127-fold when inuR gene was deleted. BlastP search revealed that the transporter displayed the highest identity to a fructose transporter protein (Frz1) from Zygosaccharomyces bailii (Pina et al. 2004). An additional putative sugar transporter gene (An15g03940) was induced on sucrose in an InuR dependant way. In contrast to the other two transporters (An15g00310 and An15g04060) this transporter is relatively highly expressed on both xylose and in the ΔinuR mutant, resulting in relative small fold-changes. An additional gene (An17g01710) was strongly induced on sucrose (about 19-fold), in an InuR dependant way. The protein encoded by An17g01710 is annotated as a multidrug resistance (MDR) protein because of its highest identity to other MDR proteins of the Major Facilitator Superfamily of transporters. Since the substrates of MDR proteins are hard to predict on the basis of the amino acid sequence, the annotation should be interpreted with care and does not exclude the possibility that this protein has a role in sugar transport. Apart from the An15g04060 transporter which might function as a fructose transporter based on the strong homology towards Frz1p (Pina et al. 2004), a useful prediction of substrates of the other putative transporters can not be made.
In the list of induced genes (Table 1), a considerable number are related to transcription, translation and amino acid metabolism. The increase in expression of those genes might indicate an overall increase of metabolic activity on sucrose compared to xylose and a subsequent higher expression of the protein transcription/translation machinery.
The genome wide analysis indicated that three extracellular inulinolytic genes and four putative sugar transporter genes were strongly induced on sucrose in an InuR dependant way. To identify possible elements in the promoter regions of these genes that could mediate InuR binding an in silico analysis of 1-kb upstream promoter sequences of these seven genes was performed. As shown in Fig. 6, optimal alignment of the promoter sequences revealed a consensus sequence (CGGN8CGG) which is present at least once in the promoter region of all genes. At least one of the triplets consistently is preceded by a T, resulting in a conserved TCGG motif in every proposed element. Within the second triplet, the conservation of the GG is consistent, but conservation of the C is not. This putative binding site of the InuR protein (CGGN8CGG) is identical to the binding site of the AmyR transcription factor which binds and induces the expression of amylolytic genes (Petersen et al. 1999). To examine whether AmyR is also involved in the regulation of inulinolytic genes, the ΔamyR mutant was constructed and analysed for growth on various carbon sources. As shown in Fig. 3, deletion of the amyR transcription factor did not result in a growth defect on inulin, sucrose or other carbon sources. The ΔamyR strain did show a strong growth reduction on maltose (data not shown). To examine a possible overlapping role of the InuR and AmyR transcription factors for inulin and/or sucrose utilization, the ΔamyR/ΔinuR double mutant was constructed. Growth analysis of the double mutant on inulin showed a minor further reduction in growth and sporulation on inulin and sucrose (Fig. 3) indicating AmyR and InuR have independent functions. Fig. 6In silico prediction of putative InuR binding sites in the promoter of sucrose induced genes. Conserved nucleotides are coloured in gray. Numbers beside the sequences indicate the position relative to the ATG of the downstream located gene
Discussion
We previously showed that the genes encoding extracellular inulinolytic enzymes in A. niger are co-regulated at the transcription level and identified sucrose, but not fructose or glucose as an efficient inducer of inulinolytic genes. It was proposed that there might be a common transcription factor to activate the expression of inulinolytic genes in response to the presence of inducing carbon sources (Yuan et al. 2006), similar to that which has been found in other carbohydrate degrading enzymes networks such as the amylolytic genes (AmyR) or the xylanolytic genes (XlnR). In this study we reported the successful identification of an inulinolytic transcriptional activator, InuR. Previous studies to isolate the AmyR and XlnR transcription factors were based on genetic screens to identify mutants with impaired expression of amylolytic and xylanolytic enzymes (Petersen et al. 1999; Gomi et al. 2000; van Peij et al. 1998a). Here, we used a different approach which made use of the full genome sequence of A. niger. The approach is based on the observation that in some cases, e.g., in the case of the AmyR transcription factor, the target genes of the transcription factor are adjacent to the transcription factor itself. In the case of the inulinolytic genes, three possible inulinolytic gene clusters were identified, each of which contains a gene encoding a putative transcription factor (Fig. 1). Disruption mutants were successfully obtained for two of the three transcription factors and subsequent phenotypic analysis indicated that only InuR (An15g00300) was involved in the inulin utilization pathway. The ΔinuR mutant showed severe reduced growth on agar plates and in submerged cultures containing sucrose or inulin as sole carbon source. Furthermore, we showed that the induction of the inulinolytic genes by inulin and sucrose is dependant on the presence of InuR. In addition to the transcription factor itself the inulinolytic gene cluster identified in A. niger consists of a sugar transporter and sucB which encodes an intracellular invertase with transfructosylating activity (Goosen et al. 2007).
To examine whether the position and clustering of the InuR orthologs was conserved among aspergilli, we compared the genes adjacent to the InuR homologous transcription factors in A. nidulans, A. oryzae and A. fumigatus. The clustering of the three genes, inuR (An15g00300), An15g00310 (putative sugar transporter) and sucB (An15g00320) in A. niger was conserved in A. nidulans (AN3835; e-value 5e-144, AN3836; e-value 7e-158 and AN3837; e-value 5e-142), and A. oryzae (AO090701000036; e-value 0.0, AO090701000037; e-value 0.0 and AO090701000038; e-value 0.0, respectively). In A. fumigatus, the InuR orthologue (Afu6g05010; e-value 0.0) is located next to the SucB orthologue (Afu6g05000; e-value 0.0), but no homolog to the putative sugar transporter (An15g00310) was present in the gene cluster in A. fumigatus. The initial gene model of the inuR gene in the A. niger annotation was lacking the ZnII(2)Cys6 binuclear cluster domain. Repositioning the intron/exon positions in N-terminal region resulted in the prediction of a 709 amino acid long protein containing a complete Zn(2)Cys6 binuclear cluster domain. BlastP searches with the A. niger InuR protein revealed orthologous genes in the three Aspergillus species examined (A. nidulans, A. oryzae and A. fumigatus). The closest homologs in A. oryzae and A. nidulans did not contain a Zn(II)2Cys6 domain and the A. fumigatus homolog only a partial Zn(II)2Cys6 cluster. The gene models of the other Aspergilli were manually inspected and by improving the gene model a Zn(II)2Cys6 domain was found to be present in the proteins. Interestingly, it turned out that the gene model of A. niger was completely conserved in the other Aspergili. Thus, the gene model of the A. nigerinuR gene could be used as a template for the prediction of the inuR gene models in the other Aspergilli. Both the numbers of introns as well as the intron/exon positions were conserved among the four Aspergilli. This result implies that gene model prediction in Aspergilli can be improved by comparison of the gene models of orthologous genes.
The inuR gene is situated in the genome next to a putative sugar transporter gene (An15g00310) and sucB. We recently showed that the sucB gene encodes an intracellular invertase with transglycosylating activity (Goosen et al. 2007). As depicted in Fig. 1, the putative sugar transporter (An15g00310) in the cluster shares the same promoter region with the sucB gene. The Northern analysis and microarray analysis showed that the two genes are not coregulated indicating that the common promoter region does not act bidirectionally as has been observed, e.g., in the case of the niiA and niaD genes (Punt et al. 1995). Expression of the gene An15g00310 was highly induced by sucrose or inulin and the induction was dependent on InuR (Fig. 5), while the sucB gene was lowly expressed on various carbon sources and hardly induced on sucrose and inulin. The function of An15g00310 remains to be elucidated but it is tempting to suggest that it involved in the transport of sucrose, or its hydrolysis products fructose or glucose.
The function of SucB in relation to inulin or sucrose metabolism is also currently unknown. We showed that deletion of the sucB did not result in a phenotype related to growth on inulin or sucrose (Goosen et al. 2007). The constitutive low level of expression of the sucB gene might suggest that SucB acts as a scouting enzyme to “sense” the presence of a useful carbon source by generating an inducer molecule which can further activate transcription of additional inulinolytic genes. A possible explanation for the lack of phenotype of the sucB deletion mutant is the presence of an addition intracellular invertase, encoded by the sucC gene. Both the enzymatic activity of the SucC protein as well as the effect of deletion of sucC and simultaneously deletion of sucB and sucC will be the subject of future research.
The genome wide transcription analysis using Affymetrix microarrays identified a limited number of genes that were strongly induced by sucrose in an InuR dependent way. These genes encode the extracellular inulinolytic enzymes and four genes encoding putative transporters. The specificity of the sugar transporters should be determined biochemically as has been done previously for A. niger sugar transporters (vanKuyk et al. 2004) since phylogenetic comparison to suggest possible substrates is not reliable (Patricia vanKuyk, personal communication). The limited number of genes that are induced by sucrose in an InuR dependant way suggests a relatively simple regulatory mechanism regarding sucrose catabolism in A. niger. Unpublished additional microarray data showed that the same three extracellular enzymes were induced on inulin in an inuR dependant way. The expression of the putative sugar transporters on inulin differed from the pattern observed on sucrose. Two of the four transporters (An17g01710 and An15g04060) were not induced on inulin after 2 and 8 h of growth on inulin. The transporter encoded by the An15g03940 gene was four to fivefold induced on inulin compared to xylose after 8 h of the transfer. Expression of this sugar transporter is also high on xylose (Table 1) and maltose, Yuan et al., unpublished results), indicating that the expression of this sugar transporter is not very tightly regulated and suggest a broad specificity of the sugar transporter. The fourth transporter (An15g00310) was more highly expressed on inulin 8 h after the transfer than on maltose and xylose (four to fivefold induction), and the induction was InuR dependant. The difference between the observed expression patterns of the extracellular enzymes and the putative sugar transporters suggest that under the different growth conditions the fungus expresses the same set of extracellular enzymes, but that the fungus fine-tunes the expression of the sugar transporters in order to allow efficient uptake. Such a fine-tuning mechanism could explain why such a large number of sugar transporters is present in the genome of A. niger as 461 proteins have been identified that belong to the Major Facilititator Superfamily of transporters (Pel et al. 2007). Such a mechanism allows the fungus to express the sugar transporters with the optimal properties in relation to substrate diversity, substrate concentration, environmental pH, and other environmental factors and nutrient conditions.
The InuR transcription factor is a member of the Zn(II)2-Cys6 DNA transcription factor family which is a fungal specific type of transcription factor. In Aspergilli, this family of transcription factors has expanded significantly when genome data is compared to that of the yeast S. cerevisiae or Neuropora crassa. In S. cerevisiae and N. crassa 54 and 89 genes, respectively (Akache et al. 2001; Borkovich et al. 2004), have been identified that contain the Zn(II)2-Cys6 DNA binding domain, the genomes of Aspergilli contain 216 (A. nidulans), 188 (A. fumigatus) or 296 (A. niger) Zn(II)2-Cys6 DNA transcription factors. The actual number of proteins containing a Zn(II)2-Cys6 DNA binding domain might still increase in Aspergilli and other fungi. We showed that the predicted gene model for the inuR gene in several Aspergilli was incorrect, resulting in a protein lacking the Zn(II)2-Cys6 DNA binding domain. Closer examination of the A. nidulans and A. niger genome annotations revealed that the difficulty of the automatic annotation in predicting the correct gene model for this class of transcription factors is not limited to inuR gene, but is a more general problem. Manual improvement of the gene models identified 326 Zn(II)2-Cys6 DNA binding domain containing proteins in A. nidulans and 480 Zn(II)2-Cys6 DNA binding domain containing proteins in A. niger (Ram and Punt, unpublished data). Thus, it is likely that other fungal genomes contain more as yet unidentified Zn(II)2-Cys6 DNA binding domain containing proteins, which could be revealed with improved gene models or manual annotation.
Transcription factors containing the Zn(II)2-Cys6 DNA binding domain usually bind the promoter region of their target genes via a conserved DNA binding site. Often, but not always, the binding site consists of conserved terminal trinucleotides, usually in symmetrical configuration (direct repeat, everted repeat or an inverted repeat) and spaced by an internal variable sequence of a defined length. Examples are the Gal4p and Lac9p transcription factors that bind to CGGN11CCG, Ppr1p and UaY that bind to CGGN6CGG, and Put3 that binds to CGGN10CCG (Todd and Andrianopoulos 1997). The binding sites for the AmyR transcription factor have been studied in detail. Several promoter regions of amylolytic genes contain a CGGN8(A/C)GG site in their promoter region to which AmyR can bind and activate transcription (Tani et al. 2001b; Ito et al. 2004).
Comparison of the 1-kb upstream promoters of the strongly InuR dependent sucrose induced genes revealed that a common consensus sequence (CGGN8CGG) is present in the promoters of all genes. Strikingly, in every putative element at least one repeat consists of a TCGG sequence (Fig. 6). This observation might be explained by a speculative, but testable, model of InuR binding and activation. We propose that InuR will bind as a dimer to the CGGN8CGG motif, and that the first InuR molecule will bind with high affinity to the TCGG DNA sequence. This binding may recruit a second InuR molecule to the site that will bind to the second motif, possibly with a weaker affinity for the DNA because of the sequence variation. Such a model for cooperative binding of a dimeric transcription factor has been experimentally substantiated for the AmyR transcription factor (Ito et al. 2004). The proposed InuR binding site (CGGN8CGG) might be conserved among filamentous fungi, as the shared promoter of the inuC and inuD genes in Penicillium sp. strain TN-88 contains a similar site (Moriyama et al. 2006).
The proposed InuR binding site is very similar to the AmyR binding site which is present in the promoter of many amylolytic genes in A. niger (Yuan et al., unpublished results) and raises immediately the question of how the specificity of the InuR and AmyR transcription factors is accomplished. Comparison of the Zn(II)2Cys6 domain of several Zn(II)2Cys6 transcription factors revealed that the Zn(II)2Cys6 domain of InuR and AmyR is not more related to each other than they are related to unrelated Zn(II)2Cys6 transcription factors (XlnR and FacB). Interestingly, the MHD of InuR and AmyR showed a high degree of identity and suggest that the MHD domain may assist in binding to the DNA target sequence. After establishing that the conserved CGGN8CGG motif in the promoter of inulinolytic genes is involved in the binding and activation of expression of the target genes, the AmyR and the InuR transcription factors are interesting subjects for further research aimed to understand how Zn(II)2-Cys6 transcription factors recognize their target genes and what determines the specificity of the binding. Analysis of the ΔamyRΔinuR double mutant indicated that both transcription factors have independent functions. The slightly decreased growth and sporulation of the ΔamyRΔinuR double mutant on sucrose compared to growth of the ΔinuR mutant on sucrose might be explained by the activity of α-glucosidases that are under control of the AmyR transcription factor. It is well established that sucrose can be hydrolyzed either by β-fructosidase, that splits sucrose by attacking the terminal, non-reducing α1, β2 linked fructose moiety, but also by an α-glucosidase catalyzing the hydrolysis of sucrose at the terminal non-reducing α1–4 linked glucose residue (Geber et al. 1992; Kelly and Kwon-Chung 1992; Alberto et al. 2004). A. niger contains several α-glucosidase encoding gene of which two of them are under control of the AmyR transcription factor. Disruption of the amyR transcription factor further decreases the level of agdA and agdB transcripts (Yuan et al., unpublished results), which might result in lower α-glucosidase activity and a subsequent decrease in the efficiency by which sucrose can be hydrolyzed.
In conclusion, we have identified InuR as the transcriptional activator required for utilization of inulin and sucrose. The results in this paper suggest that A. niger metabolizes inulin and sucrose by inducing the expression of a limited number of extracellular enzymes and sugar transporters that are under transcriptional control of a single transcription factor, InuR. The mechanism by which the InuR transcription factor is activated in response to the presence of inulin and sucrose and the mechanism that determines the specificity of the binding to the InuR target genes are the subjects of future research.
Electronic supplementary material
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Supplementary Tables (DOC 224 kb)

sucrose
transcription factor
invertase
affymetrix genechip arrays
inulinase
carbohydrate metabolism