The COMPASS (complex of proteins associated with Set1) complex represents the prototype of the SET1/MLL family of methyltransferases that controls gene transcription by H3K4 methylation (H3K4me). ...Although H2B monoubiquitination (H2Bub) is well known as a prerequisite histone mark for COMPASS activity, how H2Bub activates COMPASS remains unclear. Here, we report the cryoelectron microscopy (cryo-EM) structures of an extended COMPASS catalytic module (CM) bound to the H2Bub and free nucleosome. The COMPASS CM clamps onto the nucleosome disk-face via an extensive interface to capture the flexible H3 N-terminal tail. The interface also sandwiches a critical Set1 arginine-rich motif (ARM) that autoinhibits COMPASS. Unexpectedly, without enhancing COMPASS-nucleosome interaction, H2Bub activates the enzymatic assembly by packing against Swd1 and alleviating the inhibitory effect of the Set1 ARM upon fastening it to the acidic patch. By delineating the spatial configuration of the COMPASS-H2Bub-nucleosome assembly, our studies establish the structural framework for understanding the long-studied H2Bub-H3K4me histone modification crosstalk.
Display omitted
•Cryo-EM structures of COMPASS bound to a H2B-ubiquitinated and unmodified nucleosome•COMPASS captures nucleosomes by extensive interfacing with both histones and DNA•The Set1 ARM helix autoinhibits COMPASS upon nucleosome binding•H2Bub allosterically activates COMPASS by anchoring to the Set1 ARM helix
The cryo-EM structures of the yeast COMPASS methyltransferase bound to H2Bub and unmodified nucleosomes reveal an arginine-rich motif in the catalytic subunit, whose autoinhibitory effect on the enzymatic complex is overridden by H2B-conjugated ubiquitin. The study sheds light on the mechanism of an evolutionarily conserved crosstalk between two histone modifications.
The SET1/MLL family of histone methyltransferases is conserved in eukaryotes and regulates transcription by catalyzing histone H3K4 mono-, di-, and tri-methylation. These enzymes form a common ...five-subunit catalytic core whose assembly is critical for their basal and regulated enzymatic activities through unknown mechanisms. Here, we present the crystal structure of the intact yeast COMPASS histone methyltransferase catalytic module consisting of Swd1, Swd3, Bre2, Sdc1, and Set1. The complex is organized by Swd1, whose conserved C-terminal tail not only nucleates Swd3 and a Bre2-Sdc1 subcomplex, but also joins Set1 to construct a regulatory pocket next to the catalytic site. This inter-subunit pocket is targeted by a previously unrecognized enzyme-modulating motif in Swd3 and features a doorstop-style mechanism dictating substrate selectivity among SET1/MLL family members. By spatially mapping the functional components of COMPASS, our results provide a structural framework for understanding the multifaceted functions and regulation of the H3K4 methyltransferase family.
Display omitted
•Crystal structure of the conserved heteropentameric COMPASS catalytic core at 3.0 Å•Swd1 uses its C-terminal tail to form an inter-subunit regulatory pocket with Set1•Swd3 stimulates Set1 activity through the inter-subunit pocket via its SMART motif•A doorstop mechanism dictates substrate specificity of SET1/MLL methyltransferases
The crystal structure of the yeast COMPASS histone methyltransferase catalytic module reveals a doorstop-style mechanism dictating substrate selectivity.
When polypeptide chains fold into a protein, hydrophobic groups are compacted in the center with exclusion of water. We report the crystal structure of an alanine-rich antifreeze protein that retains ...∼400 waters in its core. The putative ice-binding residues of this dimeric, four-helix bundle protein point inwards and coordinate the interior waters into two intersecting polypentagonal networks. The bundle makes minimal protein contacts between helices, but is stabilized by anchoring to the semi-clathrate water monolayers through backbone carbonyl groups in the protein interior. The ordered waters extend outwards to the protein surface and likely are involved in ice binding. This protein fold supports both the anchored-clathrate water mechanism of antifreeze protein adsorption to ice and the water-expulsion mechanism of protein folding.
Abstract
Cleavage stimulation factor (CstF) is a highly conserved protein complex composed of three subunits that recognizes G/U-rich sequences downstream of the polyadenylation signal of eukaryotic ...mRNAs. While CstF has been identified over 25 years ago, the architecture and contribution of each subunit to RNA recognition have not been fully understood. In this study, we provide a structural basis for the recruitment of CstF-50 to CstF via interaction with CstF-77 and establish that the hexameric assembly of CstF creates a high affinity platform to target various G/U-rich sequences. We further demonstrate that CstF-77 boosts the affinity of the CstF-64 RRM to the RNA targets and CstF-50 fine tunes the ability of the complex to recognize G/U sequences of certain lengths and content.
Mammalian circadian rhythms are generated by a transcription-based feedback loop in which CLOCK:BMAL1 drives transcription of its repressors (PER1/2, CRY1/2), which ultimately interact with ...CLOCK:BMAL1 to close the feedback loop with ~24 hr periodicity. Here we pinpoint a key difference between CRY1 and CRY2 that underlies their differential strengths as transcriptional repressors. Both cryptochromes bind the BMAL1 transactivation domain similarly to sequester it from coactivators and repress CLOCK:BMAL1 activity. However, we find that CRY1 is recruited with much higher affinity to the PAS domain core of CLOCK:BMAL1, allowing it to serve as a stronger repressor that lengthens circadian period. We discovered a dynamic serine-rich loop adjacent to the secondary pocket in the photolyase homology region (PHR) domain that regulates differential binding of cryptochromes to the PAS domain core of CLOCK:BMAL1. Notably, binding of the co-repressor PER2 remodels the serine loop of CRY2, making it more CRY1-like and enhancing its affinity for CLOCK:BMAL1.
An efficient total chemical synthesis of site-specifically sumoylated histone H4 was undertaken to generate homogenously modified mononucleosomes. These were tested as substrates in biochemical ...assays with the histone H2B-specific ubiquitin ligases Rad6 and Bre1, which revealed the strong inhibition of H2B ubiquitylation by SUMO. This novel negative biochemical crosstalk between SUMO and ubiquitin was also confirmed to exist in human cells.
An efficient total chemical synthesis of site-specifically sumoylated histone H4 revealed its negative biochemical crosstalk with histone H2B ubiquitylation
in vitro
and in human cells.
OBJECTIVE:To determine graft function and survival for kidney transplants from deceased donors with acute kidney injury (AKI) that persists at the time of organ procurement.
BACKGROUND:Kidneys from ...donors with AKI are often discarded and may provide an opportunity to selectively expand the donor pool.
METHODS:Using OPTN and DonorNet data, we studied adult kidney-only recipients between 5/1/2007-12/31/2016. DonorNet was used to characterize longitudinal creatinine trends and urine output. Donor AKI was defined using KDIGO guidelines and terminal creatinine ≥ 1.5 mg/dL. We compared outcomes between AKI kidneys versus “ideal comparator” kidneys from donors with no or resolved AKI stage 1 plus terminal creatinine < 1.5 mg/dL. We fit proportional hazards models and hierarchical linear regression models for the primary outcomes of all-cause graft failure (ACGF) and 12-month estimated glomerular filtration rate (eGFR), respectively.
RESULTS:We identified 7,660 donors with persistent AKI (33.2% with AKI stage 3) from whom ≥1 kidney was transplanted. Observed rates of ACGF within 3 years were similar between recipient groups (15.5% in AKI vs 15.1% ideal comparator allografts, p = 0.2). After risk adjustment, ACGF was slightly higher among recipients of AKI kidneys (aHR 1.05, 95%CI:1.01–1.09). The mean 12-month eGFR for AKI kidney recipients was lower, but differences were not clinically important (56.6 vs. 57.5 mL/min/1.73m for ideal comparator kidneys; p < 0.001). There were 2,888 kidneys discarded from donors with AKI, age ≤ 65, without hypertension or diabetes, and terminal creatinine ≤ 4 mg/dL.
CONCLUSIONS:Kidney allografts from donors with persistent AKI are often discarded, yet those that were transplanted did not have clinically meaningful differences in graft survival and function.
BACKGROUND.Substantial differences exist in the clinical characteristics of donors across the 58 donor service areas (DSAs). Organ procurement organization (OPO) performance metrics incorporate ...organs donated after circulatory determination of death (DCDD) donors but do not measure potential DCDD donors.
METHODS.Using 2011–2016 United Network for Organ Sharing data, we examined the variability in DCDD donors/all deceased donors (%DCDD) across DSAs. We supplemented United Network for Organ Sharing data with CDC death records and OPO statistics to characterize underlying process and system factors that may correlate with donors and utilization.
RESULTS.Among 52 184 deceased donors, the %DCDD varied widely across DSAs, with a median of 15.1% (interquartile range 9.3%, 20.9%; range 0.0%–32.0%). The %DCDD had a modest positive correlation with 4 DSA factorsmedian match model for end-stage liver disease, proportion of white deaths out of total deaths, kidney center competition, and %DCDD livers by a local transplant center (all Spearman coefficients 0.289–0.464), and negative correlation with 1 factormean kidney waiting time (Spearman coefficient –0.388). Adjusting for correlated variables in linear regression explained 46.3% of the variability in %DCDD.
CONCLUSIONS.Donor pool demographics, waitlist metrics, center competition, and DCDD utilization explain only a portion of the variability of DCDD donors. This requires further studies and policy changes to encourage consideration of all possible organ donors.
Substantial differences exist in the clinical characteristics of donors across the 58 donor service areas (DSAs). Organ procurement organization (OPO) performance metrics incorporate organs donated ...after circulatory determination of death (DCDD) donors but do not measure potential DCDD donors.
Using 2011-2016 United Network for Organ Sharing data, we examined the variability in DCDD donors/all deceased donors (%DCDD) across DSAs. We supplemented United Network for Organ Sharing data with CDC death records and OPO statistics to characterize underlying process and system factors that may correlate with donors and utilization.
Among 52 184 deceased donors, the %DCDD varied widely across DSAs, with a median of 15.1% (interquartile range 9.3%, 20.9%; range 0.0%-32.0%). The %DCDD had a modest positive correlation with 4 DSA factors: median match model for end-stage liver disease, proportion of white deaths out of total deaths, kidney center competition, and %DCDD livers by a local transplant center (all Spearman coefficients 0.289-0.464), and negative correlation with 1 factor: mean kidney waiting time (Spearman coefficient -0.388). Adjusting for correlated variables in linear regression explained 46.3% of the variability in %DCDD.
Donor pool demographics, waitlist metrics, center competition, and DCDD utilization explain only a portion of the variability of DCDD donors. This requires further studies and policy changes to encourage consideration of all possible organ donors.
The post-translational modification of histones by the small ubiquitin-like modifier (SUMO) protein has been associated with gene regulation, centromeric localization, and double-strand break repair ...in eukaryotes. Although sumoylation of histone H4 was specifically associated with gene repression, this could not be proven due to the challenge of site-specifically sumoylating H4 in cells. Biochemical crosstalk between SUMO and other histone modifications, such as H4 acetylation and H3 methylation, that are associated with active genes also remains unclear. We addressed these challenges in mechanistic studies using an H4 chemically modified at Lys12 by SUMO-3 (H4K12su) and incorporated into mononucleosomes and chromatinized plasmids for functional studies. Mononucleosome-based assays revealed that H4K12su inhibits transcription-activating H4 tail acetylation by the histone acetyltransferase p300, as well as transcription-associated H3K4 methylation by the extended catalytic module of the Set1/COMPASS (complex of proteins associated with Set1) histone methyltransferase complex. Activator- and p300-dependent in vitro transcription assays with chromatinized plasmids revealed that H4K12su inhibits both H4 tail acetylation and RNA polymerase II-mediated transcription. Finally, cell-based assays with a SUMO-H4 fusion that mimics H4 tail sumoylation confirmed the negative crosstalk between histone sumoylation and acetylation/methylation. Thus, our studies establish the key role for histone sumoylation in gene silencing and its negative biochemical crosstalk with active transcription-associated marks in human cells.
PDF
