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How multicellular organisms modulate transcription in response to internal and external cues is a fundamental question in plant and animal development and growth. In Arabidopsis, TOPLESS(TPL), a Groucho/Tup1 like co-repressor, is involved in the transcriptional response to multiple plant hormones and plays a crucial role in a range of major developmental processes, such as embryonic apical-basal patterning and post-embryonic floral patterning. This dissertation describes the advances made toward the elucidation of the molecular mechanisms underlying transcriptional regulation by TPL. As a co-repressor, TPL lacks intrinsic DNA binding activity and has been shown to be recruited by transcription factors with repressive motifs. To date, a range of transcription factors have been reported to interact with TPL, yet the functionality of most of these interactions is not known. We found that the members of the Class III Homedomain-Leucine Zipper (HD- ZIPIII) transcription factor family co-localize with TPL to form complexes at the promoters of thousands of genes in the genome. HD-ZIPIIIs both antagonistically and cooperatively regulate entire pathways at multiple steps in a cell or tissue specific manner. The five members in the family form homo- and hetero-dimers and act both as activators and repressors. The association with TPL confers the repressive function of HD-ZIPIIIs. Just like its counterparts in animals and fungi, TPL also associates with histone deacetylases(HDACs) to repress transcription. We characterized the binding profiles of TPL and HDA19, a class I histone deacetylase that interacts with TPL, and found they tend to bind to the promoters of dynamically expressed genes. Almost all HDA19 peaks have an overlapping TPL peak, while about half of the TPL peaks are in common with HDA19, suggesting HDA19 is involved only at a subset of TPL targets. Specifically, we found the three members of the evolutionarily conserved NAM/CUC (for NO APICAL MERISTEM and CUP-SHA

How multicellular organisms modulate transcription in response to internal and external cues is a fundamental question in plant and animal development and growth. In Arabidopsis, TOPLESS(TPL), a Groucho/Tup1 like co-repressor, is involved in the transcriptional response to multiple plant hormones and plays a crucial role in a range of major developmental processes, such as embryonic apical-basal patterning and post-embryonic floral patterning. This dissertation describes the advances made toward the elucidation of the molecular mechanisms underlying transcriptional regulation by TPL. As a co-repressor, TPL lacks intrinsic DNA binding activity and has been shown to be recruited by transcription factors with repressive motifs. To date, a range of transcription factors have been reported to interact with TPL, yet the functionality of most of these interactions is not known. We found that the members of the Class III Homedomain-Leucine Zipper (HD- ZIPIII) transcription factor family co-localize with TPL to form complexes at the promoters of thousands of genes in the genome. HD-ZIPIIIs both antagonistically and cooperatively regulate entire pathways at multiple steps in a cell or tissue specific manner. The five members in the family form homo- and hetero-dimers and act both as activators and repressors. The association with TPL confers the repressive function of HD-ZIPIIIs. Just like its counterparts in animals and fungi, TPL also associates with histone deacetylases(HDACs) to repress transcription. We characterized the binding profiles of TPL and HDA19, a class I histone deacetylase that interacts with TPL, and found they tend to bind to the promoters of dynamically expressed genes. Almost all HDA19 peaks have an overlapping TPL peak, while about half of the TPL peaks are in common with HDA19, suggesting HDA19 is involved only at a subset of TPL targets. Specifically, we found the three members of the evolutionarily conserved NAM/CUC (for NO APICAL MERISTEM and CUP-SHA

Background:Beyond their role in post-transcriptional gene silencing, Dicer and Argonaute, two components of the RNA interference (RNAi) machinery, were shown to be involved in epigenetic regulation of centromeric heterochromatin and transcriptional gene silencing. In particular, RNAi mechanisms appear to play a role in repeat induced silencing and some aspects of Polycomb-mediated gene silencing. However, the functional interplay of RNAi mechanisms and Polycomb group (PcG) pathways at endogenous loci remains to be elucidated.Principal Findings:Here we show that the endogenous Dicer-2/Argonaute-2 RNAi pathway is dispensable for the PcG mediated silencing of the homeotic Bithorax Complex (BX-C). Although Dicer-2 depletion triggers mild transcriptional activation at Polycomb Response Elements (PREs), this does not induce transcriptional changes at PcG-repressed genes. Moreover, Dicer-2 is not needed to maintain global levels of methylation of lysine 27 of histone H3 and does not affect PRE-mediated higher order chromatin structures within the BX-C. Finally bioinformatic analysis, comparing published data sets of PcG targets with Argonaute-2-bound small RNAs reveals no enrichment of these small RNAs at promoter regions associated with PcG proteins.Conclusions:We conclude that the Dicer-2/Argonaute-2 RNAi pathway, despite its role in pairing sensitive gene silencing of transgenes, does not have a role in PcG dependent silencing of major homeotic gene cluster loci in Drosophila. © 2013 Cernilogar et al.

Transcription regulation involves enzyme-mediated changes in chromatin structure. Here, we describe a novel mode of histone crosstalk during gene silencing, in which histone H2A monoubiquitylation is coupled to the removal of histone H3 Lys 36 dimethylation (H3K36me2). This pathway was uncovered through the identification of dRING-associated factors (dRAF), a novel Polycomb group (PcG) silencing complex harboring the histone H2A ubiquitin ligase dRING, PSC and the F-box protein, and demethylase dKDM2. In vivo, dKDM2 shares many transcriptional targets with Polycomb and counteracts the histone methyltransferases TRX and ASH1. Importantly, cellular depletion and in vitro reconstitution assays revealed that dKDM2 not only mediates H3K36me2 demethylation but is also required for efficient H2A ubiquitylation by dRING/PSC. Thus, dRAF removes an active mark from histone H3 and adds a repressive one to H2A. These findings reveal coordinate trans-histone regulation by a PcG complex to mediate gene repression.

Both Caspase-3 and Caspase-9 play critical roles in the execution of mitochondria-mediated apoptosis. Caspase-9 binds to Apaf-1 in the presence of cytochrome c and dATP/ATP, and is activated by self-cleavage. Caspase-3 is activated by cleavage of caspase-8 and caspase-9. Over hundred direct caspase-3 substrates are identified whereas only few direct caspase-9 substrates are known. Here, we demonstrate that Ring1B, a component of polycomb protein complex that plays important roles in modulating chromatin structures, is a direct substrate of active caspase-3 and caspase-9 both in vitro and in vivo. The specific cleavage sites for caspase-3 and caspase-9 were mapped to Asp(175) and ASP(208), respectively. Importantly, cleavage of Ring1B by active caspases-3 and caspase-9 triggers the redistribution of Ring1B, from exclusive nuclear localization to even distribution throughout the entire cell. The transcriptional repression activity of Ring1B was also disrupted by caspase cleavage. Our data suggest that caspases-3 and caspase-9 play novel roles in transcription by regulating polycomb protein function through direct cleaving of Ring1B. (c) 2007 Elsevier B.V. All rights reserved.

Both Caspase-3 and Caspase-9 play critical roles in the execution of mitochondria-mediated apoptosis. Caspase-9 binds to Apaf-1 in the presence of cytochrome c and dATP/ATP, and is activated by self-cleavage. Caspase-3 is activated by cleavage of caspase-8 and caspase-9. Over hundred direct caspase-3 substrates are identified whereas only few direct caspase-9 substrates are known. Here, we demonstrate that Ring1B, a component of polycomb protein complex that plays important roles in modulating chromatin structures, is a direct substrate of active caspase-3 and caspase-9 both in vitro and in vivo. The specific cleavage sites for caspase-3 and caspase-9 were mapped to Asp(175) and ASP(208), respectively. Importantly, cleavage of Ring1B by active caspases-3 and caspase-9 triggers the redistribution of Ring1B, from exclusive nuclear localization to even distribution throughout the entire cell. The transcriptional repression activity of Ring1B was also disrupted by caspase cleavage. Our data suggest that caspases-3 and caspase-9 play novel roles in transcription by regulating polycomb protein function through direct cleaving of Ring1B. (c) 2007 Elsevier B.V. All rights reserved.

Both Caspase-3 and Caspase-9 play critical roles in the execution of mitochondria-mediated apoptosis. Caspase-9 binds to Apaf-1 in the presence of cytochrome c and dATP/ATP, and is activated by self-cleavage. Caspase-3 is activated by cleavage of caspase-8 and caspase-9. Over hundred direct caspase-3 substrates are identified whereas only few direct caspase-9 substrates are known. Here, we demonstrate that Ring1B, a component of polycomb protein complex that plays important roles in modulating chromatin structures, is a direct substrate of active caspase-3 and caspase-9 both in vitro and in vivo. The specific cleavage sites for caspase-3 and caspase-9 were mapped to Asp(175) and ASP(208), respectively. Importantly, cleavage of Ring1B by active caspases-3 and caspase-9 triggers the redistribution of Ring1B, from exclusive nuclear localization to even distribution throughout the entire cell. The transcriptional repression activity of Ring1B was also disrupted by caspase cleavage. Our data suggest that caspases-3 and caspase-9 play novel roles in transcription by regulating polycomb protein function through direct cleaving of Ring1B. (c) 2007 Elsevier B.V. All rights reserved.