A TLR4/TRAF6-dependent signaling pathway mediates NCoR coactivator complex formation for inflammatory gene activation.

The nuclear receptor corepressor (NCoR) forms a complex with histone deacetylase 3 (HDAC3) that mediates repressive functions of unliganded nuclear receptors and other transcriptional repressors by deacetylation of histone substrates. Recent studies provide evidence that NCoR/HDAC3 complexes can also exert coactivator functions in brown adipocytes by deacetylating and activating PPARγ coactivator 1α (PGC1α) and that signaling via receptor activator of nuclear factor kappa-B (RANK) promotes the formation of a stable NCoR/HDAC3/PGC1ß complex that coactivates nuclear factor kappa-B (NFκB)- and activator protein 1 (AP-1)-dependent genes required for osteoclast differentiation. Here, we demonstrate that activation of Toll-like receptor (TLR) 4, but not TLR3, the interleukin 4 (IL4) receptor nor the Type I interferon receptor, also promotes assembly of an NCoR/HDAC3/PGC1ß coactivator complex. Receptor-specific utilization of TNF receptor-associated factor 6 (TRAF6) and downstream activation of extracellular signal-regulated kinase 1 (ERK1) and TANK-binding kinase 1 (TBK1) accounts for the common ability of RANK and TLR4 to drive assembly of an NCoR/HDAC3/PGC1ß complex in macrophages. ERK1, the p65 component of NFκB, and the p300 histone acetyltransferase (HAT) are also components of the induced complex and are associated with local histone acetylation and transcriptional activation of TLR4-dependent enhancers and promoters. These observations identify a TLR4/TRAF6-dependent signaling pathway that converts NCoR from a corepressor of nuclear receptors to a coactivator of NFκB and AP-1 that may be relevant to functions of NCoR in other developmental and homeostatic processes.

RANK ligand converts the NCoR/HDAC3 co-repressor to a PGC1ß- and RNA-dependent co-activator of osteoclast gene expression.

The nuclear receptor co-repressor (NCoR) complex mediates transcriptional repression dependent on histone deacetylation by histone deacetylase 3 (HDAC3) as a component of the complex. Unexpectedly, we found that signaling by the receptor activator of nuclear factor κB (RANK) converts the NCoR/HDAC3 co-repressor complex to a co-activator of AP-1 and NF-κB target genes that are required for mouse osteoclast differentiation. Accordingly, the dominant function of NCoR/HDAC3 complexes in response to RANK signaling is to activate, rather than repress, gene expression. Mechanistically, RANK signaling promotes RNA-dependent interaction of the transcriptional co-activator PGC1ß with the NCoR/HDAC3 complex, resulting in the activation of PGC1ß and inhibition of HDAC3 activity for acetylated histone H3. Non-coding RNAs Dancr and Rnu12, which are associated with altered human bone homeostasis, promote NCoR/HDAC3 complex assembly and are necessary for RANKL-induced osteoclast differentiation in vitro. These findings may be prototypic for signal-dependent functions of NCoR in other biological contexts.

Perspectives on ATP-dependent chromatin remodeling.

Nucleosomes are intrinsically immobile, and thus, ATP-dependent chromatin remodeling factors are needed to alter nucleosomes to facilitate DNA-directed processes such as transcription. More generally, chromatin remodeling factors mediate chromatin dynamics, which encompasses nucleosome assembly, movement, and disruption as well as histone exchange. Here, I present selected thoughts and perspectives on the past, present, and future of these fascinating ATP-driven motor proteins.

Analysis of the Drosophila and human DPR elements reveals a distinct human variant whose specificity can be enhanced by machine learning.

The RNA polymerase II core promoter is the site of convergence of the signals that lead to the initiation of transcription. Here, we performed a comparative analysis of the downstream core promoter region (DPR) in Drosophila and humans by using machine learning. These studies revealed a distinct human-specific version of the DPR and led to the use of machine learning models for the identification of synthetic extreme DPR motifs with specificity for human transcription factors relative to Drosophila factors and vice versa. More generally, machine learning models could similarly be used to design synthetic DNA elements with customized functional properties.

NDF is a transcription factor that stimulates elongation by RNA polymerase II.

RNA polymerase II (Pol II) elongation is a critical step in gene expression. Here we found that NDF, which was identified as a bilaterian nucleosome-destabilizing factor, is also a Pol II transcription factor that stimulates elongation with plain DNA templates in the absence of nucleosomes. NDF binds directly to Pol II and enhances elongation by a different mechanism than that used by transcription factor TFIIS. Moreover, yeast Pdp3, which is related to NDF, binds to Pol II and stimulates elongation. Thus, NDF is a Pol II binding transcription elongation factor that is localized over gene bodies and is conserved from yeast to humans.

Reconstitution of Chromatin by Stepwise Salt Dialysis.

Chromatin, rather than plain DNA, is the natural substrate of the molecular machines that mediate DNA-directed processes in the nucleus. Chromatin can be reconstituted in vitro by using different methodologies. The salt dialysis method yields chromatin that consists of purified histones and DNA. This biochemically pure chromatin is well-suited for a wide range of applications. Here, we describe simple and straightforward protocols for the reconstitution of chromatin by stepwise salt dialysis and the analysis of the chromatin by the micrococcal nuclease (MNase) digestion assay. Chromatin that is reconstituted with this method can be used for efficient homology-directed repair (HDR)-mediated gene edited with the CRISPR-Cas9 system as well as for biochemical studies of chromatin dynamics and function.

Identification of the human DPR core promoter element using machine learning.

The RNA polymerase II (Pol II) core promoter is the strategic site of convergence of the signals that lead to the initiation of DNA transcription1-5, but the downstream core promoter in humans has been difficult to understand1-3. Here we analyse the human Pol II core promoter and use machine learning to generate predictive models for the downstream core promoter region (DPR) and the TATA box. We developed a method termed HARPE (high-throughput analysis of randomized promoter elements) to create hundreds of thousands of DPR (or TATA box) variants, each with known transcriptional strength. We then analysed the HARPE data by support vector regression (SVR) to provide comprehensive models for the sequence motifs, and found that the SVR-based approach is more effective than a consensus-based method for predicting transcriptional activity. These results show that the DPR is a functionally important core promoter element that is widely used in human promoters. Notably, there appears to be a duality between the DPR and the TATA box, as many promoters contain one or the other element. More broadly, these findings show that functional DNA motifs can be identified by machine learning analysis of a comprehensive set of sequence variants.

Enhancement of homology-directed repair with chromatin donor templates in cells.

A key challenge in precise genome editing is the low efficiency of homology-directed repair (HDR). Here we describe a strategy for increasing the efficiency of HDR in cells by using a chromatin donor template instead of a naked DNA donor template. The use of chromatin, which is the natural form of DNA in the nucleus, increases the frequency of HDR-edited clones as well as homozygous editing. In addition, transfection of chromatin results in negligible cytotoxicity. These findings suggest that a chromatin donor template should be useful for a wide range of HDR applications such as the precise insertion or replacement of DNA fragments that contain the coding regions of genes.

Genome editing is a powerful tool used across a wide range of biomedical research. There are several different techniques used, depending on the type of edit being made, and one known as homology-directed repair ­ or HDR for short ­ is a common technique for precisely inserting large sections of DNA, such as those needed to make desired proteins in cells. HDR takes advantage of the cell's mechanisms for repairing damage to DNA if both strands of the DNA double helix are broken. The mechanism relies on a DNA template to stitch the strands back together. To insert or replace a new DNA sequence, scientists can add a customized piece of DNA of their choosing to the cell so that it might be incorporated into the genome. However, HDR is not very efficient, and the success rate is often less than a few percent. In HDR gene editing, the DNA template is typically added as purified, or 'naked', DNA. However, the natural form of DNA in cells, known as chromatin, is where the DNA helix is wrapped around a cluster of proteins known as histones. Cruz-Becerra and Kadonaga tested the idea that DNA in the form of chromatin might be more effective as a template for HDR than naked DNA. The two approaches were compared to see which was better at inserting a sequence at three different locations in the genome of lab-grown human cells. In these experiments, the chromatin templates were 2.3- to 7.4-fold more efficient than the naked DNA. Also, the DNA in human cells is organized as pairs of chromosomes, and chromatin was better than naked DNA for editing both copies of the chromosome pairs rather than only one of them. In addition, the chromatin is potentially less toxic to the cells. Cruz-Becerra and Kadonaga hope that this will be useful for increasing the success rate of HDR experiments and potentially other methods of gene editing in the future.

The tardigrade damage suppressor protein binds to nucleosomes and protects DNA from hydroxyl radicals.

Tardigrades, also known as water bears, are animals that can survive extreme conditions. The tardigrade Ramazzottius varieornatus contains a unique nuclear protein termed Dsup, for damage suppressor, which can increase the resistance of human cells to DNA damage under conditions, such as ionizing radiation or hydrogen peroxide treatment, that generate hydroxyl radicals. Here we find that R. varieornatus Dsup is a nucleosome-binding protein that protects chromatin from hydroxyl radicals. Moreover, a Dsup ortholog from the tardigrade Hypsibius exemplaris similarly binds to nucleosomes and protects DNA from hydroxyl radicals. Strikingly, a conserved region in Dsup proteins exhibits sequence similarity to the nucleosome-binding domain of vertebrate HMGN proteins and is functionally important for nucleosome binding and hydroxyl radical protection. These findings suggest that Dsup promotes the survival of tardigrades under diverse conditions by a direct mechanism that involves binding to nucleosomes and protecting chromosomal DNA from hydroxyl radicals.

The transformation of the DNA template in RNA polymerase II transcription: a historical perspective.

The discovery of RNA polymerases I, II, and III opened up a new era in gene expression. Here I provide a personal retrospective account of the transformation of the DNA template, as it evolved from naked DNA to chromatin, in the biochemical analysis of transcription by RNA polymerase II. These studies have revealed new insights into the mechanisms by which transcription factors function with chromatin to regulate gene expression.

The RNA Polymerase II Core Promoter in Drosophila.

Transcription by RNA polymerase II initiates at the core promoter, which is sometimes referred to as the "gateway to transcription." Here, we describe the properties of the RNA polymerase II core promoter in Drosophila The core promoter is at a strategic position in the expression of genes, as it is the site of convergence of the signals that lead to transcriptional activation. Importantly, core promoters are diverse in terms of their structure and function. They are composed of various combinations of sequence motifs such as the TATA box, initiator (Inr), and downstream core promoter element (DPE). Different types of core promoters are transcribed via distinct mechanisms. Moreover, some transcriptional enhancers exhibit specificity for particular types of core promoters. These findings indicate that the core promoter is a central component of the transcriptional apparatus that regulates gene expression.

Identification of evolutionarily conserved downstream core promoter elements required for the transcriptional regulation of Fushi tarazu target genes.

The regulation of transcription initiation is critical for developmental and cellular processes. RNA polymerase II (Pol II) is recruited by the basal transcription machinery to the core promoter where Pol II initiates transcription. The core promoter encompasses the region from -40 to +40 bp relative to the +1 transcription start site (TSS). Core promoters may contain one or more core promoter motifs that confer specific properties to the core promoter, such as the TATA box, initiator (Inr) and motifs that are located downstream of the TSS, namely, motif 10 element (MTE), the downstream core promoter element (DPE) and the Bridge, a bipartite core promoter element. We had previously shown that Caudal, an enhancer-binding homeodomain transcription factor and a key regulator of the Hox gene network, is a DPE-specific activator. Interestingly, pair-rule proteins have been implicated in enhancer-promoter communication at the engrailed locus. Fushi tarazu (Ftz) is an enhancer-binding homeodomain transcription factor encoded by the ftz pair-rule gene. Ftz works in concert with its co-factor, Ftz-F1, to activate transcription. Here, we examined whether Ftz and Ftz-F1 activate transcription with a preference for a specific core promoter motif. Our analysis revealed that similarly to Caudal, Ftz and Ftz-F1 activate the promoter containing a TATA box mutation to significantly higher levels than the promoter containing a DPE mutation, thus demonstrating a preference for the DPE motif. We further discovered that Ftz target genes are enriched for a combination of functional downstream core promoter elements that are conserved among Drosophila species. Thus, the unique combination (Inr, Bridge and DPE) of functional downstream core promoter elements within Ftz target genes highlights the complexity of transcriptional regulation via the core promoter in the transcription of different developmental gene regulatory networks.

Molecular basis of chromatin remodeling by Rhp26, a yeast CSB ortholog.

CSB/ERCC6 belongs to an orphan subfamily of SWI2/SNF2-related chromatin remodelers and plays crucial roles in gene expression, DNA damage repair, and the maintenance of genome integrity. The molecular basis of chromatin remodeling by Cockayne syndrome B protein (CSB) is not well understood. Here we investigate the molecular mechanism of chromatin remodeling by Rhp26, a Schizosaccharomyces pombe CSB ortholog. The molecular basis of chromatin remodeling and nucleosomal epitope recognition by Rhp26 is distinct from that of canonical chromatin remodelers, such as imitation switch protein (ISWI). We reveal that the remodeling activities are bidirectionally regulated by CSB-specific motifs: the N-terminal leucine-latch motif and the C-terminal coupling motif. Rhp26 remodeling activities depend mainly on H4 tails and to a lesser extent on H3 tails, but not on H2A and H2B tails. Rhp26 promotes the disruption of histone cores and the release of free DNA. Finally, we dissected the distinct contributions of two Rhp26 C-terminal regions to chromatin remodeling and DNA damage repair.

NDF, a nucleosome-destabilizing factor that facilitates transcription through nucleosomes.

Our understanding of transcription by RNA polymerase II (Pol II) is limited by our knowledge of the factors that mediate this critically important process. Here we describe the identification of NDF, a nucleosome-destabilizing factor that facilitates Pol II transcription in chromatin. NDF has a PWWP motif, interacts with nucleosomes near the dyad, destabilizes nucleosomes in an ATP-independent manner, and facilitates transcription by Pol II through nucleosomes in a purified and defined transcription system as well as in cell nuclei. Upon transcriptional induction, NDF is recruited to the transcribed regions of thousands of genes and colocalizes with a subset of H3K36me3-enriched regions. Notably, the recruitment of NDF to gene bodies is accompanied by an increase in the transcript levels of many of the NDF-enriched genes. In addition, the global loss of NDF results in a decrease in the RNA levels of many genes. In humans, NDF is present at high levels in all tested tissue types, is essential in stem cells, and is frequently overexpressed in breast cancer. These findings indicate that NDF is a nucleosome-destabilizing factor that is recruited to gene bodies during transcriptional activation and facilitates Pol II transcription through nucleosomes.

A simple and versatile system for the ATP-dependent assembly of chromatin.

Chromatin is the natural form of DNA in the eukaryotic nucleus and is the substrate for diverse biological phenomena. The functional analysis of these processes ideally would be carried out with nucleosomal templates that are assembled with customized core histones, DNA sequences, and chromosomal proteins. Here we report a simple, reliable, and versatile method for the ATP-dependent assembly of evenly spaced nucleosome arrays. This minimal chromatin assembly system comprises the Drosophila nucleoplasmin-like protein (dNLP) histone chaperone, the imitation switch (ISWI) ATP-driven motor protein, core histones, template DNA, and ATP. The dNLP and ISWI components were synthesized in bacteria, and each protein could be purified in a single step by affinity chromatography. We show that the dNLP-ISWI system can be used with different DNA sequences, linear or circular DNA, bulk genomic DNA, recombinant or native Drosophila core histones, native human histones, the linker histone H1, the non-histone chromosomal protein HMGN2, and the core histone variants H3.3 and H2A.V. The dNLP-ISWI system should be accessible to a wide range of researchers and enable the assembly of customized chromatin with specifically desired DNA sequences, core histones, and other chromosomal proteins.

The punctilious RNA polymerase II core promoter.

The signals that direct the initiation of transcription ultimately converge at the core promoter, which is the gateway to transcription. Here we provide an overview of the RNA polymerase II core promoter in bilateria (bilaterally symmetric animals). The core promoter is diverse in terms of its composition and function yet is also punctilious, as it acts with strict rules and precision. We additionally describe an expanded view of the core promoter that comprises the classical DNA sequence motifs, sequence-specific DNA-binding transcription factors, chromatin signals, and DNA structure. This model may eventually lead to a more unified conceptual understanding of the core promoter.

The human initiator is a distinct and abundant element that is precisely positioned in focused core promoters.

DNA sequence signals in the core promoter, such as the initiator (Inr), direct transcription initiation by RNA polymerase II. Here we show that the human Inr has the consensus of BBCA+1BW at focused promoters in which transcription initiates at a single site or a narrow cluster of sites. The analysis of 7678 focused transcription start sites revealed 40% with a perfect match to the Inr and 16% with a single mismatch outside of the CA+1 core. TATA-like sequences are underrepresented in Inr promoters. This consensus is a key component of the DNA sequence rules that specify transcription initiation in humans.

The prenucleosome, a stable conformational isomer of the nucleosome.

Chromatin comprises nucleosomes as well as nonnucleosomal histone-DNA particles. Prenucleosomes are rapidly formed histone-DNA particles that can be converted into canonical nucleosomes by a motor protein such as ACF. Here we show that the prenucleosome is a stable conformational isomer of the nucleosome. It consists of a histone octamer associated with ∼ 80 base pair (bp) of DNA, which is located at a position that corresponds to the central 80 bp of a nucleosome core particle. Monomeric prenucleosomes with free flanking DNA do not spontaneously fold into nucleosomes but can be converted into canonical nucleosomes by an ATP-driven motor protein such as ACF or Chd1. In addition, histone H3K56, which is located at the DNA entry and exit points of a canonical nucleosome, is specifically acetylated by p300 in prenucleosomes relative to nucleosomes. Prenucleosomes assembled in vitro exhibit properties that are strikingly similar to those of nonnucleosomal histone-DNA particles in the upstream region of active promoters in vivo. These findings suggest that the prenucleosome, the only known stable conformational isomer of the nucleosome, is related to nonnucleosomal histone-DNA species in the cell.