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.

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.

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 Dmp8-Dmp18 bicistron messenger RNA enables unusual translation during cellular stress.

Alternatives to the cap mechanism in translation are often used by viruses and cells to allow them to synthesize proteins in events of stress and viral infection. In Drosophila there are hundreds of polycistronic messenger RNA (mRNA), and various mechanisms are known to achieve this. However, proteins in a same mRNA often work in the same cellular mechanism, this is not the case for Drosophila's Swc6/p18Hamlet homolog Dmp18, part of the SWR1 chromatin remodeling complex, who is encoded in a bicistronic mRNA next to Dmp8 (Dmp8-Dmp18 transcript), a structural component of transcription factor TFIIH. The organization of these two genes as a bicistron is conserved in all arthropods, however the length of the intercistronic sequence varies from more than 90 to 2 bases, suggesting an unusual translation mechanism for the second open reading frame. We found that even though translation of Dmp18 occurs independently from that of Dmp8, it is necessary for Dmp18 to be in that conformation to allow its correct translation during cellular stress caused by damage via heat-shock and UV radiation.

TFIIH localization is highly dynamic during zygotic genome activation in Drosophila, and its depletion causes catastrophic mitosis.

In Drosophila, zygotic genome activation occurs in pre-blastoderm embryos during rapid mitotic divisions. How the transcription machinery is coordinated to achieve this goal in a very brief time span is still poorly understood. Transcription factor II H (TFIIH) is fundamental for transcription initiation by RNA polymerase II (RNAPII). Herein, we show the in vivo dynamics of TFIIH at the onset of transcription in Drosophila embryos. TFIIH shows an oscillatory behaviour between the nucleus and cytoplasm. TFIIH foci are observed from interphase to metaphase, and colocalize with those for RNAPII phosphorylated at serine 5 (RNAPIIS5P) at prophase, suggesting that transcription occurs during the first mitotic phases. Furthermore, embryos with defects in subunits of either the CAK or the core subcomplexes of TFIIH show catastrophic mitosis. Although, transcriptome analyses show altered expression of several maternal genes that participate in mitosis, the global level of RNAPIIS5P in TFIIH mutant embryos is similar to that in the wild type, therefore, a direct role for TFIIH in mitosis cannot be ruled out. These results provide important insights regarding the role of a basal transcription machinery component when the zygotic genome is activated.

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.

TFIIH: New Discoveries Regarding its Mechanisms and Impact on Cancer Treatment.

The deregulation of gene expression is a characteristic of cancer cells, and malignant cells require very high levels of transcription to maintain their cancerous phenotype and survive. Therefore, components of the basal transcription machinery may be considered as targets to preferentially kill cancerous cells. TFIIH is a multisubunit basal transcription factor that also functions in nucleotide excision repair. The recent discoveries of some small molecules that interfere with TFIIH and that preferentially kill cancer cells have increased researchers' interest to elucidate the complex mechanisms by which TFIIH operates. In this review, we summarize the knowledge generated during the 25 years of TFIIH research, highlighting the recent advances in TFIIH structural and mechanistic analyses that suggest the potential of TFIIH as a target for cancer treatment.

Analysis of Drosophila p8 and p52 mutants reveals distinct roles for the maintenance of TFIIH stability and male germ cell differentiation.

Eukaryotic gene expression is activated by factors that interact within complex machinery to initiate transcription. An important component of this machinery is the DNA repair/transcription factor TFIIH. Mutations in TFIIH result in three human syndromes: xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Transcription and DNA repair defects have been linked to some clinical features of these syndromes. However, how mutations in TFIIH affect specific developmental programmes, allowing organisms to develop with particular phenotypes, is not well understood. Here, we show that mutations in the p52 and p8 subunits of TFIIH have a moderate effect on the gene expression programme in the Drosophila testis, causing germ cell differentiation arrest in meiosis, but no Polycomb enrichment at the promoter of the affected differentiation genes, supporting recent data that disagree with the current Polycomb-mediated repression model for regulating gene expression in the testis. Moreover, we found that TFIIH stability is not compromised in p8 subunit-depleted testes that show transcriptional defects, highlighting the role of p8 in transcription. Therefore, this study reveals how defects in TFIIH affect a specific cell differentiation programme and contributes to understanding the specific syndrome manifestations in TFIIH-afflicted patients.