Subunits of the Histone Chaperone CAF1 Also Mediate Assembly of Protamine-Based Chromatin

One of the most dramatic forms of chromatin reorganization occurs during spermatogenesis, when the paternal genome is repackaged from a nucleosomal to a protamine-based structure. We assessed the role of the canonical histone chaperone CAF1 in Drosophila spermatogenesis. In this process, CAF1 does not behave as a complex, but its subunits display distinct chromatin dynamics. During histone-to-protamine replacement, CAF1-p180 dissociates from the DNA while CAF1-p75 binds and stays on as a component of sperm chromatin. Association of CAF1-p75 with the paternal genome depends on CAF1-p180 and protamines. Conversely, CAF1-p75 binds protamines and is required for their incorporation into sperm chromatin. Histone removal, however, occurs independently of CAF1 or protamines. Thus, CAF1-p180 and CAF1-p75 function in a temporal hierarchy during sperm chromatin assembly, with CAF1-p75 acting as a protamine-loading factor. These results show that CAF1 subunits mediate the assembly of two fundamentally different forms of chromatin

dRYBP counteracts chromatin-dependent activation and repression of transcription

Chromatin dependent activation and repression of transcription is regulated by the histone modifying enzymatic activities of the trithorax (trxG) and Polycomb (PcG) proteins. To investigate the mechanisms underlying their mutual antagonistic activities we analyzed the function of Drosophila dRYBP, a conserved PcG- and trxG-associated protein. We show that dRYBP is itself ubiquitylated and binds ubiquitylated proteins. Additionally we show that dRYBP maintains H2A monoubiquitylation, H3K4 monomethylation and H3K36 dimethylation levels and does not affect H3K27 trimethylation levels. Further we show that dRYBP interacts with the repressive SCE and dKDM2 proteins as well as the activating dBRE1 protein. Analysis of homeotic phenotypes and post-translationally modified histones levels show that dRYBP antagonizes dKDM2 and dBRE1 functions by respectively preventing H3K36me2 demethylation and H2B monoubiquitylation. Interestingly, our results show that inactivation of dBRE1 produces trithorax-like related homeotic transformations, suggesting that dBRE1 functions in the regulation of homeotic genes expression. Our findings indicate that dRYBP regulates morphogenesis by counteracting transcriptional repression and activation. Thus, they suggest that dRYBP may participate in the epigenetic plasticity important during normal and pathological development

DOC1-Dependent Recruitment of NURD Reveals Antagonism with SWI/SNF during Epithelial-Mesenchymal Transition i

The Nucleosome Remodeling and Deacetylase (NURD) complex is a key regulator of cell differentiation that has also been implicated in tumorigenesis. Loss of the NURD subunit Deleted in Oral Cancer 1 (DOC1) is associated with human oral squamous cell carcinomas (OSCCs). Here, we show that restoration of DOC1 expression in OSCC cells leads to a reversal of epithelial-mesenchymal transition (EMT). This is caused by the DOC1-dependent targeting of NURD to repress key transcriptional regulators of EMT. NURD recruitment drives extensive epigenetic reprogramming, including eviction of the SWI/SNF remodeler, formation of inaccessible chromatin, H3K27 deacetylation, and binding of PRC2 and KDM1A, followed by H3K27 methylation and H3K4 demethylation. Strikingly, depletion of SWI/SNF mimics the effects of DOC1 re-expression. Our results suggest that SWI/SNF and NURD function antagonistically to control chromatin state and transcription. We propose that disturbance of this dynamic equilibrium may lead to defects in gene expression that promote oncogenesis

The WD-repeat protein superfamily in Arabidopsis: conservation and divergence in structure and function

BACKGROUND: The WD motif (also known as the Trp-Asp or WD40 motif) is found in a multitude of eukaryotic proteins involved in a variety of cellular processes. Where studied, repeated WD motifs act as a site for protein-protein interaction, and proteins containing WD repeats (WDRs) are known to serve as platforms for the assembly of protein complexes or mediators of transient interplay among other proteins. In the model plant Arabidopsis thaliana, members of this superfamily are increasingly being recognized as key regulators of plant-specific developmental events. RESULTS: We analyzed the predicted complement of WDR proteins from Arabidopsis, and compared this to those from budding yeast, fruit fly and human to illustrate both conservation and divergence in structure and function. This analysis identified 237 potential Arabidopsis proteins containing four or more recognizable copies of the motif. These were classified into 143 distinct families, 49 of which contained more than one Arabidopsis member. Approximately 113 of these families or individual proteins showed clear homology with WDR proteins from the other eukaryotes analyzed. Where conservation was found, it often extended across all of these organisms, suggesting that many of these proteins are linked to basic cellular mechanisms. The functional characterization of conserved WDR proteins in Arabidopsis reveals that these proteins help adapt basic mechanisms for plant-specific processes. CONCLUSIONS: Our results show that most Arabidopsis WDR proteins are strongly conserved across eukaryotes, including those that have been found to play key roles in plant-specific processes, with diversity in function conferred at least in part by divergence in upstream signaling pathways, downstream regulatory targets and /or structure outside of the WDR regions

Cytoplasmic TAF2-TAF8-TAF10 complex provides evidence for nuclear holo-TFIID assembly from preformed submodules

General transcription factor TFIID is a cornerstone of RNA polymerase II transcription initiation in eukaryotic cells. How human TFIID-a megadalton-sized multiprotein complex composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs)-assembles into a functional transcription factor is poorly understood. Here we describe a heterotrimeric TFIID subcomplex consisting of the TAF2, TAF8 and TAF10 proteins, which assembles in the cytoplasm. Using native mass spectrometry, we define the interactions between the TAFs and uncover a central role for TAF8 in nucleating the complex. X-ray crystallography reveals a non-canonical arrangement of the TAF8-TAF10 histone fold domains. TAF2 binds to multiple motifs within the TAF8 C-terminal region, and these interactions dictate TAF2 incorporation into a core-TFIID complex that exists in the nucleus. Our results provide evidence for a stepwise assembly pathway of nuclear holo-TFIID, regulated by nuclear import of preformed cytoplasmic submodules

Mathematical context in interactive documents

In this paper we introduce the concept of an interactive mathematical document. We give a formal description of such a document, which enables us to introduce the notion of a context as user and time dependent information regarding both mathematical and personal data. We also describe the realization of interactive mathematical documents within the MathDox system developed at Eindhoven University of Technology

GAGA can mediate enhancer function in trans by linking two separate DNA molecules

Enhancers have been defined as cis-acting DNA sequences that stimulate transcription from a linked promoter in a distance- and orientation-independent manner. How enhancers activate gene transcription over vast chromosomal distances within metazoan genomes remains poorly understood. Here, we show that the transcription factor GAGA can stimulate transcription by linking an enhancer to its cognate promoter. Strikingly, in addition to facilitating activation by a remote enhancer in cis, GAGA can direct activation of a promoter by an enhancer located on a separate DNA molecule. Enhancer function in trans is critically dependent on POZ domain-mediated GAGA oligomerization, enabling GAGA to bind two DNA molecules simultaneously. Transcriptional activation by an enhancer functioning in trans was observed both in transfected cells and in reconstituted transcription reactions. We propose that GAGA facilitates long-range activation by providing a protein bridge that mediates enhancer–promoter communication