Project description:Histone deacetylases (HDACs) are long recognized for important functions in regulating gene transcription in biology and diseases. However, unlike for ubiquitously expressed class I HDACs, we have little mechanistic understanding of class II HDACs that have postulated tissue specific functions. Here we report that class IIa Hdac4 and Hdac7 are up-regulated in transcription through IL-6-Stat3 signaling selectively in mouse T-helper 17 (Th17) cells during lineage-specific differentiation, but not in Th1, Th2, and Treg subtypes. Genetic knockout of Hdac4 or Hdac7 established their importance for Th17 differentiation with different functional mechanisms. Hdac4 works with Th17 transcription factors JunB-Irf4-Batf for transcriptional activation of Th17 signature genes Il17a/f, whereas Hdac7 acts distinctively in concert with Aiolos/Ncor1/Hdac3 co-repressor complex for transcriptional repression of Th17 negative regulators including Il2. Pharmacological inhibition or gene knockout of Hdac4 and Hdac7 ameliorates development of T-cell transfer induced colitis and experimental autoimmune encephalomyelitis (EAE) in mice through blockage of Th17 cell development in colon and central nervous system that recapitulate inflammatory bowel diseases and multiple sclerosis in humans, respectively. Our study highlights a previously unknown distinct mechanisms of Hdac4 and Hdac7 in transcriptional control of Th17 cell development, and rationalizes a new therapeutic strategy of targeting class IIa HDAC4/7 for the treatment of Th17-related inflammatory and autoimmune diseases.
Project description:We analyzed the genome wide distributions of HDAC1, HDAC4, HDAC7 in Th17 cells. We find that majority of HDAC4 and HDAC7 binding sites are HDAC1 bound. TMP269 inhibits HDAC4 and HDAC7 at promoter sites of Th17 negative regulator genes, leading to their upregulation through increased H3, H4 acetylation.
Project description:Our class IIa HDAC inhibitor, NVS-HD1, inhibited HDAC4 with less than 1 nM potency while exhibiting >200 fold selectivity on class IIa HDACs compared to class I (HDAC1, 3, 8) and class IIb (HDAC6) HDACs, making it the most potent and selective class IIa HDAC inhibitor reported so far. We tested the efficacy of NVS-HD1 in the mouse denervation model, either alone or on the genetic background of HDAC4 whole-body inducible knockout (HDAC4 iRKO). Global gene expression changes in gastrocnemius muscles were profiled by RNAseq. In the innervated control legs, HDAC4 knockout or NVS-HD1 caused little changes in gene expression compared to WT mice. HDAC4 knockout or NVS-HD1 mainly reversed denervation induced changes and the genes regulated by them largely overlap, suggesting that NVS-HD1 is quite specific against class IIa HDACs.
Project description:Title of Publication: Histone Deacetylase 7 Regulates Cell Survival and TCR Signaling in CD4/CD8 Double-Positive Thymocytes Abstract of publicaton: CD4/CD8 double-positive (DP) thymocytes express the transcriptional repressor Histone Deacetylase 7 (HDAC7), a class IIa HDAC that is exported from the cell nucleus after T cell receptor (TCR) engagement. Through signal-dependent nuclear export, class IIa HDACs such as HDAC7 mediate signal-dependent changes in gene expression that are important to developmental fate decisions in multiple tissues. We report that HDAC7 is exported from the cell nucleus during positive selection in thymocytes, and regulates genes mediating the coupling between TCR engagement and downstream events that determine cell survival. Thymocytes lacking HDAC7 are inefficiently positively selected due to a severely shortened lifespan and exhibit a truncated repertoire of TCR J segments. The expression of multiple important mediators and modulators of the response to TCR engagement is altered in HDAC7-deficient thymocytes, resulting in increased tonic MAP kinase activity that contributes to the observed loss of viability. Remarkably, the activity of Protein Kinase D, the kinase that mediates nuclear export of HDAC7 in response to TCR signaling, is also increased in HDAC7-deficient thymocytes, suggesting that HDAC7 nuclear export governs a self-sustaining auto-excitatory loop. These experiments add to the understanding of the life/death decision in thymic T cell development, define a novel function for class IIa HDACs, and point to a novel feed-forward mechanism whereby these molecules regulate their own state and mediate stable developmental transitions. Goal of Microarray experiment: We did these experiments to determine how alteration of the function of HDAC7, a site-specific and signal-dependent repressor of transcription, changes gene expression in CD4/CD8 DP thymocytes. Three biological replicate samples were prepared for each non-wild type mouse strain (Samples 7-18). Six biological replicates were prepared for the wild type strain (Samples 1-6). Total RNA was prepared from isolated CD4/CD8 Double-positive thymocytes and labeled with the Affymetrix Whole-Transcript labeling protocol. Labeled probes were hybridized to one Affymetrix Mouse Gene 1.0ST array each. Data from .cel files were normalized using RMA, in normalization groups representing each of the binary comparisons made. These binary comparisons between sample groups represent gene expression changes due to loss of HDAC7 (samples 1-3 vs. 7-9), transgenic expression of an HDAC7-VP16 fusion protein (samples 4-6 vs. 10-12), positive thymic selection (samples 1-6 vs. samples 11-15), and negative thymic selection (Samples 11-15 vs. samples 16-18).
Project description:Abstract of publicaton: CD4/CD8 double-positive (DP) thymocytes express the transcriptional repressor Histone Deacetylase 7 (HDAC7), a class IIa HDAC that is exported from the cell nucleus after T cell receptor (TCR) engagement. Through signal-dependent nuclear export, class IIa HDACs such as HDAC7 mediate signal-dependent changes in gene expression that are important to developmental fate decisions in multiple tissues. We report that HDAC7 is exported from the cell nucleus during positive selection in thymocytes, and regulates genes mediating the coupling between TCR engagement and downstream events that determine cell survival. Thymocytes lacking HDAC7 are inefficiently positively selected due to a severely shortened lifespan and exhibit a truncated repertoire of TCR Jalpha segments. The expression of multiple important mediators and modulators of the response to TCR engagement is altered in HDAC7-deficient thymocytes, resulting in increased tonic MAP kinase activity that contributes to the observed loss of viability. Remarkably, the activity of Protein Kinase D, the kinase that mediates nuclear export of HDAC7 in response to TCR signaling, is also increased in HDAC7-deficient thymocytes, suggesting that HDAC7 nuclear export governs a self-sustaining auto-excitatory loop. These experiments add to the understanding of the life/death decision in thymic T cell development, define a novel function for class IIa HDACs, and point to a novel feed-forward mechanism whereby these molecules regulate their own state and mediate stable developmental transitions. Title of manuscript: Nuclear Export of Histone Deacetylase 7 During Thymic Selection Mediates Immune Self-tolerance. abstract of manuscript: Histone Deacetylase 7 (HDAC7) is a TCR signal-dependent regulator of differentiation that is highly expressed in CD4/CD8 double-positive (DP) thymocytes. Here we examine the effect of blocking TCR-dependent nuclear export of HDAC7 during thymic selection, through expression of a signal-resistant mutant of HDAC7 (HDAC7-?P) in thymocytes. We find that HDAC7-?P Transgenic thymocytes exhibit a profound block in negative thymic selection, but can still undergo positive selection, resulting in the escape of autoreactive T cells into the periphery. Gene expression profiling reveals a comprehensive suppression of the negative selection-associated gene expression program in DP thymocytes, associated with a defect in the activation of MAP kinase pathways by TCR signals. The consequence of this block in vivo is a lethal autoimmune syndrome involving the exocrine pancreas and other abdominal organs. These experiments establish a novel molecular model of autoimmunity and cast new light on the relationship between thymic selection and immune self-tolerance. Goal of Microarray experiment: We did these experiments to determine how alteration of the function of HDAC7, a site-specific and signal-dependent repressor of transcription, changes gene expression in CD4/CD8 DP thymocytes.
Project description:Analysis of Class II Histone Deacetylase (HDAC) regulation of hepatic gluconeogenesis at the gene expression level. We show that in liver, Class IIa HDACs (HDAC4, 5, and 7) are all phosphorylated and excluded from the nucleus by AMPK family kinases. In response to the fasting hormone glucagon, Class IIa HDACs rapidly translocate to the nucleus where they directly bind to the promoters of gluconeogenic enzymes such as G6Pase. In turn, HDAC4/5 mediate the acute transcriptional induction of these genes via deacetylation and activation of Foxo family transcription factors. Loss of Class IIa HDACs in the murine liver results in inhibition of FOXO target genes and lowers blood glucose, resulting in increased glycogen storage. Total RNA obtained from primary hepatocytes infected with shGFP or shHDAC4 & 5 subjected to 2 or 4 hours treatment with DMSO or forskolin.
Project description:Reversible protein acetylation provides a central mechanism for controlling gene expression and cellular signaling events. It is governed by the antagonistic commitment of two enzymes families: the histone acetyltransferases (HATs) and the histone deacetylases (HDACs). HDAC4, like its class IIa counterparts, is a potent transcriptional repressor through interactions with tissue-specific transcription factors via its N-terminal domain. Whilst the lysine deacetylase activity of the class IIa HDACs is much less potent than that of the class I enzymes, HDAC4 has been reported to influence protein deacetylation through its interaction with HDAC3. To investigate the influence of HDAC4 on protein acetylation, we employed the unbiased AcetylScan proteomic screen. We identified many proteins known to be modified by acetylation, but found that the absence of HDAC4 had no effect on the acetylation profile of the murine neonate brain. This is consistent with the biochemical data suggesting that HDAC4 may not function as a lysine deacetylase, but these in vivo data do not support the previous report showing that the enzymatic activity of HDAC3 might be modified by its interaction with HDAC4. To complement this work, we used Affymetrix arrays to investigate the effect of HDAC4 knock-out on the transcriptional profile of the postnatal murine brain. There was no effect on global transcription, consistent with the absence of a differential histone acetylation profile. Validation of the array data by Taq-man qPCR indicated that only protamine 1 and Igfbp6 mRNA levels were increased by more than one-fold and only CamK4 was decreased. The lack of a major effect on the transcriptional profile is consistent with the cytoplasmic location of HDAC4 in the P3 murine brain. mRNA expression analysis was performed by microarray in 3-day-old HDAC4 KO pups and WT littermates. Ten samples were analysed for each genotype. Microarray quality control was performed using the software package provided on RACE (http://race.unil.ch).
Project description:Analysis of Class II Histone Deacetylase (HDAC) regulation of hepatic gluconeogenesis at the gene expression level. We show that in liver, Class IIa HDACs (HDAC4, 5, and 7) are all phosphorylated and excluded from the nucleus by AMPK family kinases. In response to the fasting hormone glucagon, Class IIa HDACs rapidly translocate to the nucleus where they directly bind to the promoters of gluconeogenic enzymes such as G6Pase. In turn, HDAC4/5 mediate the acute transcriptional induction of these genes via deacetylation and activation of Foxo family transcription factors. Loss of Class IIa HDACs in the murine liver results in inhibition of FOXO target genes and lowers blood glucose, resulting in increased glycogen storage.
Project description:Reversible protein acetylation provides a central mechanism for controlling gene expression and cellular signaling events. It is governed by the antagonistic commitment of two enzymes families: the histone acetyltransferases (HATs) and the histone deacetylases (HDACs). HDAC4, like its class IIa counterparts, is a potent transcriptional repressor through interactions with tissue-specific transcription factors via its N-terminal domain. Whilst the lysine deacetylase activity of the class IIa HDACs is much less potent than that of the class I enzymes, HDAC4 has been reported to influence protein deacetylation through its interaction with HDAC3. To investigate the influence of HDAC4 on protein acetylation, we employed the unbiased AcetylScan proteomic screen. We identified many proteins known to be modified by acetylation, but found that the absence of HDAC4 had no effect on the acetylation profile of the murine neonate brain. This is consistent with the biochemical data suggesting that HDAC4 may not function as a lysine deacetylase, but these in vivo data do not support the previous report showing that the enzymatic activity of HDAC3 might be modified by its interaction with HDAC4. To complement this work, we used Affymetrix arrays to investigate the effect of HDAC4 knock-out on the transcriptional profile of the postnatal murine brain. There was no effect on global transcription, consistent with the absence of a differential histone acetylation profile. Validation of the array data by Taq-man qPCR indicated that only protamine 1 and Igfbp6 mRNA levels were increased by more than one-fold and only CamK4 was decreased. The lack of a major effect on the transcriptional profile is consistent with the cytoplasmic location of HDAC4 in the P3 murine brain.
Project description:B lymphopoiesis is the result of several cell lineage choices and differentiation steps whose perturbation leads to B cell malignancies. Cellular transitions for B cell generation have been associated with gene activation and silencing by networks of B cell specific transcription factors (TFs) and dynamic changes in DNA methylation. How gene repression is established and which lineage-specific transcriptional repressors are involved during B cell lymphopoiesis are still not totally understood. Here, by using our in vivo experimental approach, we have found that HDAC7 represses Tet2 enzyme in pro-B cells. In fact, HDAC7 deficient pro-B cells show a significant increase in the percentage of global 5-hydroxymethylation. To prove the role of HDAC7 in 5-hydroxymethylation, we have performed a genome-wide experimental approach. hMeDIP-sequencing experiments reveal an increase in the enrichment of this epigenetic modification at many loci related to lineage inappropriate genes. Our results corroborate that HDAC7 is an essential transcriptional regulator during early B cell development that silences lineage or functionally inappropriate genes and unveil an unexpected role of a class IIa HDAC in controlling DNA methylation