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Gene network transitions in embryos and other fate-changing contexts involve combinations of transcription factors. A subset of fate-changing transcription factors act as pioneers; they scan and target nucleosomal DNA and initiate cooperative events that can open the local chromatin. However, a gap has remained in understanding how molecular interactions with the nucleosome contribute to the chromatin-opening phenomenon. Here we identified a short α-helical region, conserved among FOXA pioneer factors, that interacts with core histones and contributes to chromatin opening in vitro. The same domain is involved in chromatin opening in early mouse embryos for normal development. Thus, local opening of chromatin by interactions between pioneer factors and core histones promotes genetic programming.
Diabetes mellitus arises from insufficient insulin secretion from pancreatic islet β-cells. In type 2 diabetes (T2D), β-cell dysfunction is associated with inactivation and/or loss of transcription factor (TF) activity, including Pdx1. Notably, this particular TF is viewed as a master regulator of pancreas development and islet β-cell formation, identity and function. TFs, like Pdx1, recruit coregulators to transduce activating and/or repressing signals to the general transcriptional machinery for controlling gene expression, including modifiers of DNA, histones and nucleosome architecture. These coregulators impart a secondary layer of control that can be exploited to modulate TF activity. In this review, we describe Pdx1-recruited coregulators that impact chromatin structure, consequently influencing normal β-cell function and likely Pdx1 activity in pathophysiological settings.
© 2016 John Wiley & Sons Ltd.
It is demonstrated that time-dependent density functional theory (DFT) calculations can accurately predict changes in near-UV electronic circular dichroism (ECD) spectra of DNA as the structure is altered from the linear (free) B-DNA form to the supercoiled N-DNA form found in nucleosome core particles. At the DFT/B3LYP level of theory, the ECD signal response is reduced by a factor of 6.7 in going from the B-DNA to the N-DNA form, and it is illustrated how more than 90% of the individual base-pair dimers contribute to this strong hypochromic effect. Of the several inter-base pair parameters, an increase in twist angles is identified as to strongly contribute to a reduced ellipticity. The present work provides first evidence that first-principles calculations can elucidate changes in DNA dichroism due to the supramolecular organization of the nucleoprotein particle and associates these changes with the local structural features of nucleosomal DNA.
Lipid electrophiles modify cellular targets, altering their function. Here, we describe histones as major targets for modification by 4-oxo-2-nonenal, resulting in a stable Lys modification structurally analogous to other histone Lys acylations. Seven adducts were identified in chromatin isolated from intact cells: four 4-ketoamides to Lys and three Michael adducts to His. A 4-ketoamide adduct residing at H3K27 was identified in stimulated macrophages. Modification of histones H3 and H4 prevented nucleosome assembly.
To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation.
Histone lysine methylation is a dynamic process that plays an important role in regulating chromatin structure and gene expression. Recent studies have identified Jhd2, a JmjC domain-containing protein, as an H3K4-specific demethylase in budding yeast. However, important questions regarding the regulation and functions of Jhd2 remain unanswered. In this study, we show that Jhd2 has intrinsic activity to remove all three states of H3K4 methylation in vivo and can dynamically associate with chromatin to modulate H3K4 methylation levels on both active and repressed genes and at the telomeric regions. We found that the plant homeodomain (PHD) finger of Jhd2 is important for its chromatin association in vivo. However, this association is not dependent on H3K4 methylation and the H3 N-terminal tail, suggesting the presence of an alternative mechanism by which Jhd2 binds nucleosomes. We also provide evidence that the JmjN domain and its interaction with the JmjC catalytic domain are important for Jhd2 function and that Not4 (an E3 ligase) monitors the structural integrity of this interdomain interaction to maintain the overall protein levels of Jhd2. We show that the S451R mutation in human SMCX (a homolog of Jhd2), which has been linked to mental retardation, and the homologous T359R mutation in Jhd2 affect the protein stability of both of these proteins. Therefore, our findings provide a mechanistic explanation for the observed defects in patients harboring this SMCX mutant and suggest the presence of a conserved pathway involving Not4 that modulates the protein stability of both yeast Jhd2 and human SMCX.
The mechanism by which ubiquitination of histone H2B (H2Bub1) regulates H3-K4 and -K79 methylation and the histone H2A-H2B chaperone Spt16-mediated nucleosome dynamics during transcription is not fully understood. Upon investigating the effect of H2Bub1 on chromatin structure, we find that contrary to the supposed role for H2Bub1 in opening up chromatin, it is important for nucleosome stability. First, we show that H2Bub1 does not function as a "wedge" to non-specifically unfold chromatin, as replacement of ubiquitin with a bulkier SUMO molecule conjugated to the C-terminal helix of H2B cannot functionally support H3-K4 and -K79 methylation. Second, using a series of biochemical analyses, we demonstrate that nucleosome stability is reduced or enhanced, when the levels of H2Bub1 are abolished or increased, respectively. Besides transcription elongation, we show that H2Bub1 regulates initiation by stabilizing nucleosomes positioned over the promoters of repressed genes. Collectively, our study reveals an intrinsic difference in the property of chromatin assembled in the presence or absence of H2Bub1 and implicates the regulation of nucleosome stability as the mechanism by which H2Bub1 modulates nucleosome dynamics and histone methylation during transcription.
Regulation of eukaryotic gene expression is far more complex than one might have imagined 30 years ago. However, progress towards understanding gene regulatory mechanisms has been rapid and comprehensive, which has made the integration of detailed observations into broadly connected concepts a challenge. This review attempts to integrate the following concepts: (1) a well-defined organization of nucleosomes and modification states at most genes; (2) regulatory networks of sequence-specific transcription factors; (3) chromatin remodeling coupled to promoter assembly of the general transcription factors and RNA polymerase II; and (4) phosphorylation states of RNA polymerase II coupled to chromatin modification states during transcription. The wealth of new insights arising from the tools of biochemistry, genomics, cell biology, and genetics is providing a remarkable view into the mechanics of gene regulation.
The predominant organizational theme by which the transcription machinery and chromatin regulators are positioned within promoter regions or throughout genes in a genome is largely unknown. We mapped the genomic location of diverse representative components of the gene regulatory machinery in Saccharomyces cerevisiae to an experimental resolution of <40 bp. Sequence-specific gene regulators, chromatin regulators, mediator, and RNA polymerase (Pol) II were found primarily near the downstream border from the "-1" nucleosome, which abuts against the approximately 140-bp nucleosome-free promoter region (NFR). General transcription factors TFIIA, -B, -D, -E, -F, -H were located near the downstream edge from the NFR. The -1 nucleosome dissociated upon Pol II recruitment, but not upon recruitment of only TBP and TFIIB. The position of many sequence-specific regulators in promoter regions correlated with the position of specific remodeling complexes, potentially reflecting functional interactions. Taken together the findings suggest that the combined action of activators and chromatin remodeling complexes remove the -1 nucleosome after the preinitiation complex (PIC) has partially assembled, but before or concomitant with Pol II recruitment. We find PIC assembly, which includes Pol II recruitment, to be a significant rate-limiting step during transcription, but that additional gene-specific rate-limiting steps associated with Pol II occur after recruitment.
Most nucleosomes are well-organized at the 5' ends of S. cerevisiae genes where "-1" and "+1" nucleosomes bracket a nucleosome-free promoter region (NFR). How nucleosomal organization is specified by the genome is less clear. Here we establish and inter-relate rules governing genomic nucleosome organization by sequencing DNA from more than one million immunopurified S. cerevisiae nucleosomes (displayed at http://atlas.bx.psu.edu/). Evidence is presented that the organization of nucleosomes throughout genes is largely a consequence of statistical packing principles. The genomic sequence specifies the location of the -1 and +1 nucleosomes. The +1 nucleosome forms a barrier against which nucleosomes are packed, resulting in uniform positioning, which decays at farther distances from the barrier. We present evidence for a novel 3' NFR that is present at >95% of all genes. 3' NFRs may be important for transcription termination and anti-sense initiation. We present a high-resolution genome-wide map of TFIIB locations that implicates 3' NFRs in gene looping.