Peer review of: The MiDAC histone deacetylase complex is essential for embryonic development and has a unique (2020)

Reviewers' comments: 
Reviewer #1 (Remarks to the Author): 

 
This manuscript reports a comprehensive functional and structural investigation of the MiDAC 
complex, the less studied of the class I HDAC complexes. The data presented convey important 
biological information, which should be of interest to a large audience. 

However before publication the authors should pay attention to the following points.  

1-Figure 1B shows that the DNTTIP1 immunoprecipitation brings down much less HDAC1 than 
MIDEAS immunoprecipitation. However, Figures 1C and D show that these two 
immunoprecipitations present comparable HDAC activities. 

This discrepancy needs to be explained. Is the immunoprecipitation shown in Figure 1B 
representative of all IPs (including those shown in C and D)? 


2-Figure 1F and 1G, it would have been more informative if the authors had shown the total 
distribution of cells along the cell cycle (i. e., FACS profiles) rather than time points (Fig. 1F) or 
indicating the % of cells only in one phase (Fig. 1G). Additionally, in Fig. 1G, the immunoprecipitated 
Flag-MIDEAS should be shown. This control is important since the total amount of DNTTIP1 in 
different phases of the cell cycle shown in Fig. 1F appears different from the co-immunoprecipitated 
DNTTIP1 shown in Fig. 1G. Indeed Co-immunoprecipitated DNTTIP1 appears more abundant in S and 
G1 than in G2/M (Fig. 1G), compared to the asynchronous cells or cells in G2/M. 


3-Figure 1H, based on the data shown, the authors propose that the HDAC activity of the complex is 
higher in G1/S than the activity recovered from both asynchronous cells or in cells in mitosis. 

However, in order to support this conclusion, they should have visualized the immunoprecipitated 
HDACs. Indeed, considering the possible variability in HDAC IP (Fig. 1B), the results could simply 
reflect the variable amounts of immunoprecipitated HDACs in the different cell types. 


4-From Supp. Figure 2D we can see that the knock-down of DNTTIP1 and MIDEAS variably affects the 
total HDAC1 protein levels. In fact, these variations at the protein levels are somehow reminiscent of 
variations at the transcript levels shown in Supp. Figure 1C. 

Would it be possible that HDAC1 gene expression is positively regulated by the complex? 

Could the authors complete these data by using their transcriptomic data (Fig. 4)? 


5-Legend Fig 2 E-F, Please define what the authors mean by “proteins were isolated from nuclear 
extracts”. Also the first line an “i” is missing in “wth”. 

Page 8 top, “A” is missing. Should be Figure 3 A and B. 

6-Figure 3 D the arrows are barely visible. Please change their size and colour. 


7-The data shown in Supp. Figure 4 are somehow inconsistently presented for MIDEAS-del and 
DNTTPA1-del cells. Please present the same information for cells of both genotypes (genotyping, 
HDAC activity, cell growth, cell cycle distribution and westerns).
 

8-Where is the principal component analysis (PCA) described at the bottom of page 8 shown? 

 

Reviewer #2 (Remarks to the Author): 

The deacetylases HDAC1 and HDAC2 are subunits of multiprotein complexes. The association with 
components of these complexes is required for their activation, regulation and recruitment. The 
MiDAC complex was identified in a chemoproteomic approach as mitosis-specific HDAC1/HDAC2 
complex but near to nothing is known about the function of this complex. In the first part of the 
paper the authors investigate the MiDAC proteins DNTTIP1 and MIDEAS. DNTTIP1, MIDEAS and 
HDAC1 are localized in the nucleus but are excluded from mitotic chromosomes. Knock-down of the 
proteins in human tumor cells caused the misalignment of mitotic chromosomes suggesting a role of 
the MiDAC complex in cell cycle regulation and maintenance of genomic integrity. Interestingly, the 
expression of both proteins is mutually co-dependent. DNTTIP1 and MIDEAS interact with each other 
throughout the cell cycle and are continuously associated with HDAC activity. In the second part of 
the manuscript the authors performed a CRISPR-Cas9-mediated knockout of DNTTIP1 and MIDEAS in 
mice. DNTTIP1 and MIDEAS KO mice die during late embryogenesis and have very similar 
phenotypes namely heart malformation and hematopoietic failure. Similar to the knock-down tumor 
cells DNTTIP1 and MIDEAS KO MEFs showed misalignment of mitotic chromosomes. However, 
proliferation and cell cycle progression was normal. Gene expression profiling revealed a similar 
deregulation of genes in both KO cell lines suggesting a potential role of the MiDAC complex in 
transcription control. Finally, the authors determined the cryo-EM structure of the MiDAC complex, 
a tetrameric structure with the four HDAC1 enzymes positioned at the periphery with outward-
facing active sites. 

Criticisms: 

In this manuscript the authors report interesting findings concerning the structure of the MiDAC 
complex and the phenotype of cells or mice lacking this complex. However, they do not provide 
insight into the mechanistic function of the MiDAC complex in mammalian cells to connect structural 
data to the mitotic phenotype or the developmental phenotype. For instance, histone 
hyperacetylation has been shown to disrupt centromeric heterochromatin and to affect kinetochore 
assembly and sister chromatid separation. Therefore, it would be important to investigate whether 
loss of MiDAC results in changes in histone acetylation and repressive histone methylation marks 
during mitosis in general and specifically at certain chromosomal regions such as centromeres. Given 
that the MiDAC complex could target multiple nucleosomes ChIP-seq experiments should be 
performed to examine whether MiDAC associates with specific chromosomal regions in the context 
of the mitotic phenotype or specific genes in the context of the deregulated genes. 

In the current form the analysis of the KO mice adds little to the manuscript. The phenotype of MEFs 
is similar to the one of knock-down U2OS cells and the impact on mitosis does not affect early 
embryogenesis. The effects on heart development and the hematopoietic system are unclear and 
might be investigated in another study. In this context, loss of the MiDAC complex could be partially 
compensated in the early phase of development by a related HDAC complex, which might be absent 
in specific differentiated cell types. 

In the first part the authors provide convincing data showing that the MiDAC complex is functional 
as deacetylase complex not only during mitosis but throughout the cell cycle. As discussed by the 
authors this is in contrast to previous findings and would suggest that the MiDAC complex has a 
more general function. Therefore it would be important to show that HDAC1 is associated with 
DNTTIP1 and MIDEAS during interphase. 

 

Reviewer #3 (Remarks to the Author): 

MiDAC is one of the class I histone deacetylases. The global structure and detailed mechanisms 
remain unclear. As other class I histone deacetylases, MiDAC is a multi-protein complex, composed 
of an HDAC catalytic subunit and two scaffold subunits, MIDEAS and DNTTIP1. In this manuscript, 
Turnbull., et al. designed a series of functional and structural studies to characterize the MiDAC 
complex and explore its functions. They determined the cryo-EM structures of MiDAC at resolutions 
of 4-5 angstrom, revealing a unique architecture different from other class I histone deacetylases. 
Overall this work was well executed with a large body of data from a combination of different tools, 
including cell biology, animal model and structural biology. Before formal acceptance for publication, 
several issues should be addressed in the revision. 

 
1. As stated in the last sentence in Abstract, “Four copies of HDAC1 are positioned at the periphery 
with outward-facing active sites so as to target multiple nucleosomes suggesting…”. “target multiple 
nucleosomes” is a hypothesis based on cryo-EM structures and not supported by any functional data 
in previous or this study. Therefore, the wording should also be more accurate. 

2. In Figure 2A, the definition of y-axis, “Relative Density”, should be explained in legend. The 
reference value for normalization in the histogram was not clearly defined. Were the column heights 
normalized based on the Control lane? If so, why the height of Control lane in the top panel was 
more than 2? In addition, in the bottom panel, the protein concentrations in MID_si1/2 were 
significantly less than Control lane, but the column heights are similar. 

3. The description in the second paragraph of Page 6. In Supplementary Figure 2A-B, the knock-down 
of MIDEAS or DNTTIP1 only suppressed the mRNA expression of their own. But in Figure 2A, the 
protein levels of both MIDEAS and DNTTIP1 were affected in either MIDEAS or DNTTIP1 depleting 
cell lines. It seems that the “mRNA expression” and “protein levels” were detected in the same 
manner using western blotting and the difference above accounted for the difference sampling: 
whole cell extract v.s. soluble nuclear fraction. This should be clarified in the revision. 

4. In Figure 3C-D, the background and contrast in these images seemed to have been manually 
adjusted. If the adjustment is necessary, details should be provided in the legend. 

5. What does the “c. 7 Å” in the first paragraph of Page 10 and other parts of the article mean? 
Please describe it upon the first usage of this term. 

6. The structural data show a unique architecture for the MiDAC complex. However, many 
conclusions in the text were presented without supporting figures provided (listed below). The 
authors should provide a number of necessary supporting figures for better presentation of their 
structural data: 

(1) The last sentence in Page 10, “Density for the InsP6, but not the SAHA, was clearly visible in the 
map.”; 

(2) The second paragraph in Page 11, “The shorter helix in MIDEAS mediates a tight interaction with 
a non-polar groove between the end of the long helices of the DNTTIP1 dimer”; 

“The SANT domains of MIDEAS and MTA1 are very similar in structure to each other and to the SANT 
domain of the SMRT protein that binds to HDAC32”; 

“One of the regions of MIDEAS most similar to other ELM2-SANT domain corepressor proteins is the 
ELM2 specific motif which binds in an extended conformation in a conserved groove on HDAC1”. 

(3) The first paragraph in Page 12, “The arrangement is such that the DNTTIP1 dimerisation domains 
are symmetrically juxtaposed, but do not directly interact.”; 
“The two S-shapes are in opposite orientations such that the four HDAC subunits are positioned at 
the periphery, resulting in a striking three-dimensional X-shaped complex (Supplementary Figure 
7H)”. “opposite orientations” should be clearly displayed. 

(4) The second paragraph in Page 12, “The tetramerisation interface between the two dimers is 
mediated by interactions between the SANT domains”; 

“Residues Lys849, Tyr874 and Gln877 contribute to the interface between the two neighbouring 
SANT domains”; 

“These residues are not conserved in MTA1”; 

“Interestingly, Lys839 and Lys843 appear to be in a position to interact with the inositol phosphate 
that is bound to the neighbouring dimer, although there is no apparent density for the side chains”; 

“This may explain why it was only possible to obtain a high-resolution map of the tetramer in the 
presence of inositol phosphate (7 Å vs 4.5 Å)”. The image processing for “the tetramer in without 
inositol phosphate” should be described in Method session. Related figure should also be provided. 

“A further obvious effect of the InsP6 was to significantly improve the local resolution of the core of 
the HDAC1, consistent with the proposed mechanism of activation”. 

(5) The third paragraph in Page 12, “In addition to the interactions between the SANT domains it 
appears that the residues Nterminal to the DNTTIP1 dimerisation domain are crossing over to 
interact with the neighbouring tetramer”. 

(6) The last paragraph in Page 12, “We also analysed cryo-EM grids of complexes containing full-
length DNTTIP1 including the DNA-binding domain (Supplementary Figure 7A-D)”. The cited figures 
were incorrect. 

(7) The first paragraph in Page 16, “Interestingly, the tetrameric arrangement brings the inositol 
phosphate binding sites on the two dimers in close proximity such that two conserved basic residues 
in the SANT domain of one MIDEAS are able to interact across the tetramerisation interface with the 
inositol phosphate in the adjacent dimer”. 

7. In Supplementary Figure 5, cryo-EM maps of the intermediate processing steps, especially for the 
results of 3D classification, should be displayed here. 

8. Judged by Supplementary Figure 6D, there seems to be a preferred orientation problem for the 
tetramer complex. Please provide a figure panel displaying the angular distribution of the final 
refinement. 

9. The Data collection and Image processing for the data from grafix cross-linking, and gentle cross-
linking samples without DNTTIP1 truncation could be included in Method section. 

10. As stated in the last paragraph of Page 11, “The most ordered regions are the DNTTIP1 
dimerisation domain and the core of HDAC1 (Figure 7A & B and Supplementary Figure 6)”. That 
means the regions of MIDEAS are less ordered? Since MIDEAS is located between DNTTIP1 and 
HDAC1, not the most peripheral, does the lower resolution correlate with its functional dynamics for 
the complex? Have the authors tried to perform local 3D classification focused on the MIDEAS region 
to separate different conformations?