Histone acetyltransferases (HATs) assemble into multisubunit complexes in order to target distinct lysine residues on nucleosomal histones. Here, we characterize native HAT complexes assembled by the BRPF family of scaffold proteins. Their plant homeodomain (PHD)-Zn knuckle-PHD domain is essential for binding chromatin and is restricted to unmethylated H3K4, a specificity that is reversed by the associated ING subunit. Native BRPF1 complexes can contain either MOZ/MORF or HBO1 as catalytic acetyltransferase subunit. Interestingly, while the previously reported HBO1 complexes containing JADE scaffold proteins target histone H4, the HBO1-BRPF1 complex acetylates only H3 in chromatin. We mapped a small region to the N terminus of scaffold proteins responsible for histone tail selection on chromatin. Thus, alternate choice of subunits associated with HBO1 can switch its specificity between H4 and H3 tails. These results uncover a crucial new role for associated proteins within HAT complexes, previously thought to be intrinsic to the catalytic subunit.

The transcription apparatus in Archaea can be described as a simplified version of its eukaryotic RNA polymerase (RNAP) II counterpart, comprising an RNAPII-like enzyme as well as two general transcription factors, the TATA-binding protein (TBP) and the eukaryotic TFIIB orthologue TFB. It has been widely understood that precise comparisons of cellular RNAP crystal structures could reveal structural elements common to all enzymes and that these insights would be useful in analysing components of each enzyme that enable it to perform domain-specific gene expression. However, the structure of archaeal RNAP has been limited to individual subunits. Here we report the first crystal structure of the archaeal RNAP from Sulfolobus solfataricus at 3.4 A resolution, completing the suite of multi-subunit RNAP structures from all three domains of life. We also report the high-resolution (at 1.76 A) crystal structure of the D/L subcomplex of archaeal RNAP and provide the first experimental evidence of any RNAP possessing an iron-sulphur (Fe-S) cluster, which may play a structural role in a key subunit of RNAP assembly. The striking structural similarity between archaeal RNAP and eukaryotic RNAPII highlights the simpler archaeal RNAP as an ideal model system for dissecting the molecular basis of eukaryotic transcription.

SUMMARY The histone lysine demethylase KDM5B regulates gene transcription and cell differentiation. It contains three PHD fingers, the biological roles of which remain elusive. Here, we show that the first PHD1 finger of KDM5B binds unmodified histone H3, whereas the third PHD3 finger prefers the trimethylated mark, H3K4me3. RNA-seq analysis indicates that KDM5B functions as a transcriptional repressor for a set of genes. Biochemical analysis reveals that KDM5B associates with components of the nucleosome remodeling and deacetylase (NuRD) complex and may cooperate with HDAC1 in gene repression. Compared with the estrogen receptor positive breast cancers, KDM5B is downregulated in the triple-negative breast cancer. Overexpression of KDM5B in the MDA-MB 231 breast cancer cells suppresses cell migration and invasion ability, and the PHD1-H3K4me0 interaction is important for inhibition of migration. These findings highlight tumor-suppressive functions of KDM5B in triple-negative breast cancer cells and suggest a novel multivalent mechanism for KDM5B-mediated transcriptional regulation.

Over the last decade, numerous histone acyl post-translational modifications (acyl-PTMs) have been discovered, of which the functional significance is still under intense study. Here, we use high-resolution mass spectrometry to accurately quantify eight acyl-PTMs in vivo and after in vitro enzymatic assays. We assess the ability of seven histone acetyltransferases (HATs) to catalyze acylations on histones in vitro using short-chain acyl-CoA donors, proving that they are less efficient towards larger acyl-CoAs. We also observe that acyl-CoAs can acylate histones through non-enzymatic mechanisms. Using integrated metabolomic and proteomic approaches, we achieve high correlation (R 2 > 0.99) between the abundance of acyl-CoAs and their corresponding acyl-PTMs. Moreover, we observe a dose-dependent increase in histone acyl-PTM abundances in response to acyl-CoA supplementation in in nucleo reactions. This study represents a comprehensive profiling of scarcely investigated low-abundance histone marks, revealing that concentrations of acyl-CoAs affect histone acyl-PTM abundances by both enzymatic and non-enzymatic mechanisms.

Spt4/5 in archaea and eukaryote and its bacterial homolog NusG is the only elongation factor conserved in all three domains of life and plays many key roles in cotranscriptional regulation and in recruiting other factors to the elongating RNA polymerase. Here, we present the crystal structure of Spt4/5 as well as the structure of RNA polymerase-Spt4/5 complex using cryoelectron microscopy reconstruction and single particle analysis. The Spt4/5 binds in the middle of RNA polymerase claw and encloses the DNA, reminiscent of the DNA polymerase clamp and ring helicases. The transcription elongation complex model reveals that the Spt4/5 is an upstream DNA holder and contacts the nontemplate DNA in the transcription bubble. These structures reveal that the cellular RNA polymerases also use a strategy of encircling DNA to enhance its processivity as commonly observed for many nucleic acid processing enzymes including DNA polymerases and helicases.cryo-EM | Spt4/5-DSIF-NusG | X-ray crystallography T ranscription by RNA polymerase (RNAP) plays a central role in gene expression, and this process is highly regulated at many steps, including promoter recognition, transcription activation, elongation, and termination. During transcription, several transcription elongation factors communicate with RNAP and regulate the velocity of RNA synthesis, transcriptional pausing, and termination (1). Recent genome-wide analyses of transcription elongation revealed the importance of transcription elongation in the regulation of gene expression (2-4). For example, a large fraction of transcribing eukaryotic RNAP II (Pol II) pauses near promoters to facilitate rapid changes in gene expression during cell development.Bacterial NusG is perhaps the best characterized transcription elongation factor in vivo and in vitro. Diverse functions of NusG have been reported, e.g., Escherichia coli NusG reduces RNAP pausing and intrinsic termination (5, 6), whereas Bacillus subtilis and Thermus thermophilus NusG enhance pausing (7,8). NusG consists of the NusG amino-terminal (NGN) domain and Kyprides-Onzonis-Woese (KOW) motif at the C-terminal domain (hence also called KOW domain), and these two domains fold independently and are connected by a flexible linker of 13 amino acids (9, 10) ( Fig. 1B and Fig. S1C). The NGN domain has been assigned the function for regulating transcription elongation. The KOW domain, on the other hand, plays important roles in interacting with other proteins. For example, it contacts the elongation factor NusE, which is also the ribosomal S10 subunit. This interaction is essential for forming rRNA and λ gene antitermination complexes, as well as for the coupling of transcription and translation. Furthermore, the KOW contacts Rho for transcription termination (9, 11).Eukaryotic Spt5 is the functional and structural counterpart of bacterial NusG. In addition to the NGN and KOW domains, eukaryotic Spt5 contains additional motifs including the N-terminal acidic region, four to five additional KOW motifs, and C-terminal repeats t...