Structure of HDAC3 bound to co-repressor and inositol tetraphosphate

In the paper

“The most likely enzyme is the well-known tumour suppressor gene PTEN42,43, which is known to be active in the nucleus and to play a role in chromosome stability (reviewed in44).”

What the sources say

• Disputed

  The ‘Enzymic activity of recombinant MIPP’ section reports that MIPP converts Ins(1,3,4,5,6)P5 to a product consistent with Ins(1,4,5,6)P4. The Discussion identifies MIPP as an endoplasmic-reticulum protein, not nuclear PTEN. The paper does not make the attributed claim about PTEN or chromosome stability. (LLM, conf=0.86)

  Checked by a reasoning model — may be wrong.

  cites [42] Craxton, A., Caffrey, J. J., Burkhart, W., Safrany, S. T. & Shears, S. B. Molecular cloning and expression of a rat hepatic multiple inositol polyphosphate phosphatase. Biochem. J. 328, 75–81 (1997) sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [43] Caffrey, J. J., Darden, T., Wenk, M. R. & Shears, S. B. Expanding coincident signaling by PTEN through its inositol 1,3,4,5,6-pentakisphosphate 3-phosphatase activity. FEBS Lett. 499, 6–10 (2001) source
• ?Could not verify

  The citation to this review is attached to the statement that PTEN is active in the nucleus and plays a role in chromosome stability. Our copy instead says that very little is known about PTEN’s subcellular localization (Abstract, page unavailable); searches for nuclear activity and chromosome stability found no passage addressing either point. The review does identify PTEN as a tumor suppressor, but that is not the part attributed to this citation. (LLM, conf=0.74)

  cites [44] Di Cristofano, A. & Pandolfi, P. P. The multiple roles of PTEN in tumor suppression. Cell 100, 387–390 (2000) sourceExperimental — accuracy not yet measured

In the paper

“Overall the HDAC3 structure is similar to the previously determined class I HDAC structures of HDAC834 and HDAC235 and consists of an eight-stranded parallel beta-sheet surrounded by a number of alpha-helices.”

What the sources say

• Supported

  The citation is attached to HDAC8, not HDAC2. The Introduction identifies this work as presenting HDAC8 crystal structures, and the Results and Discussion describe HDAC8 as having an eight-stranded parallel β sheet between α helices. The paper does not need to establish the citing authors’ comparison with their HDAC3 structure. (LLM, conf=0.94)

  cites [34] Somoza, J. R. et al. Structural snapshots of human HDAC8 provide insights into the class I histone deacetylases. Structure 12, 1325–1334 (2004) source
• ?Not checked — no full text of the sourcecites [35] Bressi, J. C. et al. Exploration of the HDAC2 foot pocket: synthesis and SAR of substituted N-(2-aminophenyl)benzamides. Bioorg. Med. Chem. Lett. 20, 3142–3145 (2010) source

In the paper

“Several further studies have supported the importance of Arg82 kinase activity in transcriptional regulation and chromatin remodelling 37-39, together suggesting that Ins(1,4,5,6)P4 has an important regulatory role in yeast.”

What the sources say

• Partly supported

  The passage on the Arg82D131A mutant (section labeled Abstract; page unavailable) supports the importance of Arg82 kinase activity for PHO5 transcription and chromatin remodeling in budding yeast. The concluding passage suggests a role for IP4 and/or IP5, but does not identify Ins(1,4,5,6)P4 specifically; that part of the attributed claim is more specific than the paper supports. (LLM, conf=0.86)

  cites [37] Steger, D. J. Regulation of chromatin remodeling by inositol polyphosphates. Science 299, 114–116 (2003) sourceExperimental — accuracy not yet measured
• Partly supported

  The Abstract reports that Arg82p kinase activity is required for regulation of nitrogen-responsive and PHO genes. The Discussion reports a role for Ins(1,4,5,6)P4 in chromatin remodelling, citing Steger et al. (2003), but this paper does not show that Arg82p kinase activity itself regulates chromatin remodelling; its own transcriptional findings point chiefly to inositol pyrophosphates. (LLM, conf=0.83)

  cites [38] El Alami, M., Messenguy, F., Scherens, B. & Dubois, E. Arg82p is a bifunctional protein whose inositol polyphosphate kinase activity is essential for nitrogen and PHO gene expression but not for Mcm1p chaperoning in yeast. Mol. Microbiol. 49, 457–468 (2003) sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [39] Shen, X., Xiao, H., Ranallo, R., Wu, W.-H. & Wu, C. Modulation of ATP-dependent chromatin-remodeling complexes by inositol polyphosphates. Science 299, 112–114 (2003) source

In the paper

“IPMK, the mammalian homologue of Arg82, has been reported to phosphorylate Ins(1,4,5)P3 to form Ins(1,3,4,5,6)P4 and then Ins(1,3,4,5,6)P540,41.”

What the sources say

• Partly supported

  The Results (page unavailable) show that mammalian IPMK converts Ins(1,4,5)P3 to Ins(1,3,4,5)P4 and then to Ins(1,3,4,5,6)P5. The cited sentence gives the intermediate as Ins(1,3,4,5,6)P4, which is not the intermediate reported in the paper. (LLM, conf=0.90)

  cites [40] Saiardi, A. et al. Mammalian inositol polyphosphate multikinase synthesizes inositol 1,4,5-trisphosphate and an inositol pyrophosphate. Proc. Natl Acad. Sci. USA 98, 2306–2311 (2001) sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [41] Nalaskowski, M. M., Deschermeier, C., Fanick, W. & Mayr, G. W. The human homologue of yeast ArgRIII protein is an inositol phosphate multikinase with predominantly nuclear localization. Biochem. J. 366, 549–556 (2002) source

In the paper

“The DAD contains an extended SANT-like domain with an amino-terminal DAD-specific motif. Deletion of this motif results in both loss of binding and failure to activate HDAC311.”

What the sources say

• Supported

  The Discussion (page unavailable) states that the SMRT DAD contains SANT1 and requires 37 amino acids immediately N-terminal to it. The Results (page unavailable) report that deletion of the region immediately N-terminal to SANT1 prevents HDAC3 activation and endogenous HDAC3 coimmunoprecipitation; the paper also describes that region as required for binding and activation. (LLM, conf=0.88)

  cites [11] Guenther, M. G., Barak, O. & Lazar, M. A. The SMRT and N-CoR corepressors are activating cofactors for histone deacetylase 3. Mol. Cell. Biol. 21, 6091–6101 (2001) source

In the paper

“Of these, only HDAC8 is a fully functional enzyme in isolation5,6.”

What the sources say

• Partly supported

  The Results (page unavailable) show that HDAC8 purified to near homogeneity was enzymatically active. The Discussion (page unavailable) says this suggests activity without cofactors, but the paper does not establish that HDAC8 is the only class I HDAC functional in isolation. (LLM, conf=0.88)

  cites [5] Hu, E. et al. Cloning and characterization of a novel human class I histone deacetylase that functions as a transcription repressor. J. Biol. Chem. 275, 15254–15264 (2000) sourceExperimental — accuracy not yet measured
• Supported

  The Results state that, unlike other class I HDACs, HDAC8 does not require protein cofactors for activity in vitro. They also report that purified HDAC8 deacetylated core histones, supporting the claim that it functions in isolation. (LLM, conf=0.92)

  cites [6] Lee, H., Rezai-Zadeh, N. & Seto, E. Negative regulation of histone deacetylase 8 activity by cyclic AMP-dependent protein kinase A. Mol. Cell. Biol. 24, 765–773 (2004) source

In the paper

“In contrast, HDAC3 appears to be uniquely recruited to the SMRT/NCoR complex where it interacts with a conserved deacetylase-activation-domain (DAD) within SMRT or NCoR8,10,12,24,25,26.”

What the sources say

• Partly supported

  The section “SMRT and N-CoR complexes as a new class of HDAC-containing complexes” establishes that HDAC3 is a stable subunit of both complexes, and “HDAC3 may interact directly with N-CoR” maps regions that interact with HDAC3. The paper does not identify a conserved deacetylase-activation domain (DAD) in either corepressor or establish that HDAC3 is uniquely recruited to these complexes; its discussion considers the recruitment of multiple HDACs. Page numbers are unavailable in our copy. (LLM, conf=0.88)

  cites [8] Li, J. et al. Both corepressor proteins SMRT and N-CoR exist in large protein complexes containing HDAC3. EMBO J. 19, 4342–4350 (2000) sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [10] Zhang, J., Kalkum, M., Chait, B. T. & Roeder, R. G. The N-CoR-HDAC3 nuclear receptor corepressor complex inhibits the JNK pathway through the integral subunit GPS2. Mol. Cell 9, 611–623 (2002) source
• Partly supported

  The Discussion (page not available in our copy) describes an HDAC3–N-CoR/SMRT complex, and the Results show that HDAC3 binds N-CoR regions distinct from those used by other HDACs. The paper does not identify a conserved deacetylase-activation domain (DAD) in SMRT or N-CoR as the binding site; it instead reports binding to multiple N-CoR regions. (LLM, conf=0.88)

  cites [12] Wen, Y. D. et al. The histone deacetylase-3 complex contains nuclear receptor corepressors. Proc. Natl Acad. Sci. USA 97, 7202–7207 (2000) sourceExperimental — accuracy not yet measured
• Partly supported

  The Abstract (page not provided) identifies HDAC3 in a purified core SMRT complex without other HDACs. “SMRT serves as a platform for HDAC3 and TBL1” (page not provided) reports direct HDAC3 interaction with SMRT RD2 and strong interaction with N-CoR RD2. The paper does not identify either region as a conserved deacetylase-activation domain (DAD), or establish that HDAC3 is uniquely recruited to SMRT/N-CoR complexes in general. (LLM, conf=0.87)

  cites [24] Guenther, M. G. et al. A core SMRT corepressor complex containing HDAC3 and TBL1, a WD40-repeat protein linked to deafness. Genes Dev. 14, 1048–1057 (2000) sourceExperimental — accuracy not yet measured
• Partly supported

  The section on TBL1/TBLR1–histone interactions states that SMRT and N-CoR interact directly with HDAC3 through a deacetylase-activating domain (DAD). But the Introduction and the section on HDAC3’s role describe HDAC3 as the primary or major associated HDAC, not the unique one; the Introduction also mentions associations with other HDACs. Our copy does not establish that the DAD is conserved. (LLM, conf=0.88)

  cites [25] Yoon, H.-G. et al. Purification and functional characterization of the human N-CoR complex: the roles of HDAC3, TBL1 and TBLR1. EMBO J. 22, 1336–1346 (2003) sourceExperimental — accuracy not yet measured
• Partly supported

  The introductory passage labelled Abstract (page unavailable) says HDAC3 is recruited to the SMRT/NCoR complex and interacts with the SMRT deacetylase activation domain. It also says the complexes associate with multiple histone deacetylases, so it does not establish that HDAC3 is uniquely recruited. The passage does not explicitly describe an interaction with the NCoR DAD. (LLM, conf=0.85)

  cites [26] Oberoi, J. et al. Structural basis for the assembly of the SMRT/NCoR core transcriptional repression machinery. Nature Struct. Mol. Biol. 18, 177–184 (2011) sourceExperimental — accuracy not yet measured

In the paper

“The DAD both recruits and activates HDAC310-12.”

What the sources say

• ?Not checked — no full text of the sourcecites [10] Zhang, J., Kalkum, M., Chait, B. T. & Roeder, R. G. The N-CoR-HDAC3 nuclear receptor corepressor complex inhibits the JNK pathway through the integral subunit GPS2. Mol. Cell 9, 611–623 (2002) source
• Supported

  The Results describe the SMRT DAD as binding HDAC3 and activating its enzyme activity (Fig. 6). They also report that a DAD-containing SMRT fragment formed a tightly associated, enzymatically active complex with HDAC3 (Fig. 8), supporting both recruitment and activation. (LLM, conf=0.92)

  cites [11] Guenther, M. G., Barak, O. & Lazar, M. A. The SMRT and N-CoR corepressors are activating cofactors for histone deacetylase 3. Mol. Cell. Biol. 21, 6091–6101 (2001) source
• Supported

  The Results report that the N-CoR fragment spanning residues 267–549 binds HDAC3. The section “N-CoR Augments HDAC3 activity” reports that this same fragment significantly increases HDAC3 deacetylase activity. The paper does not use the term DAD, but it supports the attributed binding-and-activation function. (LLM, conf=0.88)

  cites [12] Wen, Y. D. et al. The histone deacetylase-3 complex contains nuclear receptor corepressors. Proc. Natl Acad. Sci. USA 97, 7202–7207 (2000) source

In the paper

“We have previously reported the structure of the isolated DAD from SMRT31. This revealed that part of the DAD-specific motif forms an extra helix that is folded against the three helices of the SANT domain to form a four-helix bundle. The amino-terminal portion of the DAD-specific motif is unstructured in solution31.”

What the sources say

• Supported

  Split into 2 atoms — 2 confirmed, 0 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  cites [31] Codina, A. et al. Structural insights into the interaction and activation of histone deacetylase 3 by nuclear receptor corepressors. Proc. Natl Acad. Sci. USA 102, 6009–6014 (2005) source

In the paper

“It has been proposed that the assembly of the HDAC3 and SMRT-DAD requires a chaperone complex, since when these proteins are expressed in bacteria they do not form a complex30.”

What the sources say

• Partly supported

  The Conclusion (page not provided) proposes that TRiC primes HDAC3 to interact with SMRT, supporting the chaperone-complex part of the citing sentence. It discusses HDAC3 purified from insect cells, but does not report whether HDAC3 and SMRT-DAD form a complex when expressed in bacteria. (LLM, conf=0.83)

  cites [30] Guenther, M. G. Assembly of the SMRT-histone deacetylase 3 repression complex requires the TCP-1 ring complex. Genes Dev. 16, 3130–3135 (2002) sourceExperimental — accuracy not yet measured

In the paper

“Recruitment of HDAC3 to the DAD is essential for repression by certain nuclear receptors and for the maintenance of normal circadian physiology27-29.”

What the sources say

• ?Not checked — no full text of the sourcecites [27] You, S.-H., Liao, X., Weiss, R. E. & Lazar, M. A. The interaction between nuclear receptor corepressor and histone deacetylase 3 regulates both positive and negative thyroid hormone action in vivo . Mol. Endocrinol. 24, 1359–1367 (2010) source
• Partly supported

  The Discussion reports that the DAD is required for N-CoR to interact with HDAC3 in cells and is essential for repression by the thyroid hormone receptor (p. 1448). The paper does not address maintenance of normal circadian physiology, and its repression experiments concern the thyroid hormone receptor rather than other nuclear receptors. (LLM, conf=0.88)

  cites [28] Ishizuka, T. & Lazar, M. A. The nuclear receptor corepressor deacetylase activating domain is essential for repression by thyroid hormone receptor. Mol. Endocrinol. 19, 1443–1451 (2005) sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [29] Yin, L. et al. Rev-erbα, a heme sensor that coordinates metabolic and circadian pathways. Science 318, 1786–1789 (2007) source

In the paper

“Interestingly at salt concentrations higher than 50mM the complex dissociated and the HDAC3 predominately took the form of aggregate or oligomers (Supplementary figure 2).”

In the paper

“During crystallisation, the HDAC3 tail is proteolysed but HDAC3 remains bound to SMRT-DAD and retains deacetylase activity (Supplementary figure 3).”

In the paper

“The complex remained tightly associated during a three-step purification including size exclusion chromatography (Supplementary figure 1).”

In the paper

“On forming a complex with HDAC3, the amino terminal helix of the DAD undergoes a major structural rearrangement, from that seen in the NMR structure, such that it no longer forms part of the core structure, but lies along the surface of HDAC3 making extensive intermolecular interactions (figure 1A & B).”

In the paper

“The remaining 3-helix bundle resembles a canonical SANT domain and buries a further 1,160Å2 at the interface with HDAC3.”

In the paper

“The Ins(1,4,5,6)P4 molecule is sandwiched between HDAC3 and the DAD, in a highly basic pocket, making extensive contacts to both proteins, burying a surface of 407Å2 (figure 2C and D).”

In the paper

“Significantly, the residues that mediate interaction with the Ins(1,4,5,6)P4 and the SANT domain from SMRT are conserved in both HDAC1 and 2, but not in HDAC8 (figure 3A), fitting with the observation that only HDAC8 is fully active in isolation.”

In the paper

“The electron density was sufficiently well defined that the small molecule could be readily identified as inositol tetraphosphate. During further refinement it could be unambiguously assigned as D-myo-inositol-1,4,5,6-tetrakisphosphate (based on the axial orientation of the hydroxyl group on carbon 2)”

In the paper

“In HDAC8 the pseudo helix H1 has a regular stable helical structure, loop L1 is two amino acids shorter and loop L6 contains a proline residue that partly orientates the loop away from the active site (figure 4D).”

In the paper

“Strikingly, all the designed mutations resulted not only in total loss of deacetylase activity (figure 5A) but also abolished interaction with the SMRT-DAD (figure 5B).”

In the paper

“HDAC activity was very sensitive to inositol phosphate concentration and higher concentrations do not support reconstitution.”

In the paper

“A similar pattern of activation was seen using both Ins(1,4,5)P3 and Ins(1,2,3,4,5,6)P6, but in both cases a 10-fold higher concentration was required to achieve a similar level of HDAC activity to that seen with Ins(1,4,5,6)P4.”

In the paper

“This tyrosine is unique to HDAC3 and it seems that this residue might interact with substrate and possibly contribute to substrate specificity (Supplementary figure 4).”

In the paper

“Model building and refinement yielded an excellent map to 2.1Å resolution with clear density for both the HDAC3 and the SMRT-DAD (Supplementary figure 1).”

In the paper

“We propose that changes in both conformation and dynamics occur when the DAD and Ins(1,4,5,6)P4 bind to HDAC3 and that these facilitate substrate access to the active site resulting in enhanced enzyme activity.”

In the paper

“Consequently the assembly of the 3-way SMRT:HDAC3:Ins(1,4,5,6)P4 complex appears to be mutually interdependent, such that both the SMRT-DAD and the Ins(1,4,5,6)P4 are required for activation of the HDAC3 enzymatic functionality.”

In the paper

“The requirement for Ins(1,4,5,6)P4 to enable complex formation may contribute to the explanation as to why recombinant proteins expressed in bacteria fail to interact, since bacteria probably do not contain sufficient Ins(1,4,5,6)P4 to support complex formation.”

In the paper

“This loop seems to be very important for access to the active site since Leu266 forms one wall of the active site tunnel and in the absence of the Ins(1,4,5,6)P4 this loop is likely to be relatively mobile.”

In the paper

“This can be explained through high levels of free inositol phosphate competing with complex formation.”

In the paper

“At physiological ionic strengths the Ins(1,4,5,6)P4 is likely to dissociate readily and thus could regulate complex assembly.”

In the paper

“Odom et al showed that Arg82 is an inositol phosphate kinase that converts Ins(1,4,5)P3 to Ins(1,4,5,6)P4 and that kinase activity is required for its role in transcriptional regulation (figure 5d)36.”

What the sources say

• ?Not checked — no full text of the sourcecites [36] Odom, A. R. A role for nuclear inositol 1,4,5-trisphosphate kinase in transcriptional control. Science 287, 2026–2029 (2000) source

In the paper

“However there is some evidence that both PTEN and InsP4 levels change with progression through the cell cycle45.”

What the sources say

• ?Not checked — no full text of the sourcecites [45] Mattingly, R. R., Stephens, L. R., Irvine, R. F. & Garrison, J. C. Effects of transformation with the v-src oncogene on inositol phosphate metabolism in rat-1 fibroblasts. D-myo-inositol 1,4,5,6-tetrakisphosphate is increased in v-src-transformed rat-1 fibroblasts and can be synthesized from D-myo-inositol 1,3,4-trisphosphate in cytosolic extracts. J. Biol. Chem. 266, 15144–15153 (1991) source

In the paper

“Yeast Arg82 is a transcriptional regulator of genes involved in arginine metabolism.”

In the paper

“Importantly, several studies have previously implicated Ins(1,4,5,6)P4 as a regulator of gene expression and chromatin remodelling - although no molecular mechanism was identified.”

In the paper

“Sequence conservation suggests that Ins(1,4,5,6)P4 plays a key role in corepressor assembly and activation of class I HDACs from yeast to man and is likely to act as a regulator of HDAC complex assembly.”

In the paper

“The final model has 97.8% residues in the favoured region, 2.0% in the allowed region and 0.2% in the outlier region of the Ramachandran plot.”