Disordered protein regions partner up the cBAF remodeler for a chromatin dance.

Intrinsically disordered protein regions form condensates and mediate interactions with factors that regulate gene activity. Patil et al.1 decode how such regions within the chromatin remodeler cBAF choreograph self-condensation and non-self interactions with transcriptional regulators, potentially impacting disease.

A triskelion of nucleic acids drives protein aggregation in A-T.

Mutations in ataxia telangiectasia mutated (ATM) kinase lead to cerebellar neurodegeneration. In this issue of Molecular Cell, Lee et al. (2021) revealed how transcription-induced reactive oxygen species and DNA-RNA hybrids activate PARP enzymes, generating the nucleic acid poly-ADP-ribose, which promotes the accumulation of protein aggregates in A-T-like disorders.

Structures of Drosophila cryptochrome and mouse cryptochrome1 provide insight into circadian function.

Drosophila cryptochrome (dCRY) is a FAD-dependent circadian photoreceptor, whereas mammalian cryptochromes (CRY1/2) are integral clock components that repress mCLOCK/mBMAL1-dependent transcription. We report crystal structures of full-length dCRY, a dCRY loop deletion construct, and the photolyase homology region of mouse CRY1 (mCRY1). Our dCRY structures depict Phe534 of the regulatory tail in the same location as the photolesion in DNA-repairing photolyases and reveal that the sulfur loop and tail residue Cys523 plays key roles in the dCRY photoreaction. Our mCRY1 structure visualizes previously characterized mutations, an NLS, and MAPK and AMPK phosphorylation sites. We show that the FAD and antenna chromophore-binding regions, a predicted coiled-coil helix, the C-terminal lid, and charged surfaces are involved in FAD-independent mPER2 and FBXL3 binding and mCLOCK/mBMAL1 transcriptional repression. The structure of a mammalian cryptochrome1 protein may catalyze the development of CRY chemical probes and the design of therapeutic metabolic modulators.

PARP-1 Flips the Epigenetic Switch on Obesity.

PARP enzymes are increasingly taking on important roles beyond DNA repair. Huang et al. (2020b) report how the NAD+-dependent ADP-ribosylation of histone H2B by PARP-1 in complex with a metabolic enzyme suppresses the phosphorylation of an adjacent residue, impacting adipogenesis.

The Oncogenic Helicase ALC1 Regulates PARP Inhibitor Potency by Trapping PARP2 at DNA Breaks.

The anti-tumor potency of poly(ADP-ribose) polymerase (PARP) inhibitors (PARPis) has been linked to trapping of PARP1 on damaged chromatin. However, little is known about their impact on PARP2, an isoform with overlapping functions at DNA lesions. Whether the release of PARP1/2 from DNA lesions is actively catalyzed by molecular machines is also not known. We found that PARPis robustly trap PARP2 and that the helicase ALC1 (CHD1L) is strictly required for PARP2 release. Catalytic inactivation of ALC1 quantitatively traps PARP2 but not PARP1. ALC1 manipulation impacts the response to single-strand DNA breaks through PARP2 trapping, potentiates PARPi-induced cancer cell killing, and mediates synthetic lethality upon BRCA deficiency. The chromatin remodeler ALC1 actively drives PARP2 turnover from DNA lesions, and PARP2 contributes to the cellular responses of PARPi. This suggests that disrupting the ATP-fueled remodeling forces of ALC1 might enable therapies that selectively target the DNA repair functions of PARPs in cancer.

The Chaperone FACT and Histone H2B Ubiquitination Maintain S. pombe Genome Architecture through Genic and Subtelomeric Functions.

The histone chaperone FACT and histone H2B ubiquitination (H2Bub) facilitate RNA polymerase II (Pol II) passage through chromatin, yet it is not clear how they cooperate mechanistically. We used genomics, genetic, biochemical, and microscopic approaches to dissect their interplay in Schizosaccharomyces pombe. We show that FACT and H2Bub globally repress antisense transcripts near the 5' end of genes and inside gene bodies, respectively. The accumulation of these transcripts is accompanied by changes at genic nucleosomes and Pol II redistribution. H2Bub is required for FACT activity in genic regions. In the H2Bub mutant, FACT binding to chromatin is altered and its association with histones is stabilized, which leads to the reduction of genic nucleosomes. Interestingly, FACT depletion globally restores nucleosomes in the H2Bub mutant. Moreover, in the absence of Pob3, the FACT Spt16 subunit controls the 3' end of genes. Furthermore, FACT maintains nucleosomes in subtelomeric regions, which is crucial for their compaction.

A Poly-ADP-Ribose Trigger Releases the Auto-Inhibition of a Chromatin Remodeling Oncogene.

DNA damage triggers chromatin remodeling by mechanisms that are poorly understood. The oncogene and chromatin remodeler ALC1/CHD1L massively decompacts chromatin in vivo yet is inactive prior to DNA-damage-mediated PARP1 induction. We show that the interaction of the ALC1 macrodomain with the ATPase module mediates auto-inhibition. PARP1 activation suppresses this inhibitory interaction. Crucially, release from auto-inhibition requires a poly-ADP-ribose (PAR) binding macrodomain. We identify tri-ADP-ribose as a potent PAR-mimic and synthetic allosteric effector that abrogates ATPase-macrodomain interactions, promotes an ungated conformation, and activates the remodeler's ATPase. ALC1 fragments lacking the regulatory macrodomain relax chromatin in vivo without requiring PARP1 activation. Further, the ATPase restricts the macrodomain's interaction with PARP1 under non-DNA damage conditions. Somatic cancer mutants disrupt ALC1's auto-inhibition and activate chromatin remodeling. Our data show that the NAD+-metabolite and nucleic acid PAR triggers ALC1 to drive chromatin relaxation. Modular allostery in this oncogene tightly controls its robust, DNA-damage-dependent activation.

Synthesis and Macrodomain Binding of Gln-carba-ADPr-peptide.

Mono-ADP-ribosylation is a dynamic post-translational modification (PTM) with important roles in cell signalling. This modification occurs on a wide variety of amino acids, and one of the canonical modification sites within proteins is the side chain of glutamic acid. Given the transient nature of this modification (acylal linkage) and the high sensitivity of ADP-ribosylated glutamic acid, stabilized isosteres are required for structural and biochemical studies. Here, we report the synthesis of a mimic of ADP-ribosylated peptide derived from histone H2B that contains carba-ADP-ribosylated glutamine as a potential mimic for Glu-ADPr. We synthesized a cyclopentitol-ribofuranosyl derivative of 5'-phosphoribosylated Fmoc-glutamine and used this in the solid-phase synthesis of the carba-ADPr-peptide mimicking the ADP-ribosylated N-terminal tail of histone H2B. Binding studies with isothermal calorimetry demonstrate that the macrodomains of human MacroD2 and TARG1 bind to carba-ADPr-peptide in the same way as ADPr-peptides containing the native ADP-riboside moiety connected to the side chain of glutamine in the same peptide sequence.

Chromatin places metabolism center stage.

Dynamic changes in histone and transcription factor acetylation modulate gene expression. A study in Science (Wellen et al., 2009) reports that changes in glucose metabolism alter the availability of acetyl-CoA, the essential cofactor for protein acetylation. These findings reveal a direct connection between central metabolism and mammalian gene expression.

Nick Your DNA, Mark Your Chromatin.

DNA damage induces chemical and structural changes in our chromatin-embedded genome. In a recent issue of Nature Communications, Grundy et al. (2016) identify a role for PARP3 in the repair of single-strand breaks and reveal that PARP3 mono-ADP-ribosylates nucleosomal histone H2B.

ICON 2020-International Scientific Tendinopathy Symposium Consensus: the development of a core outcome set for gluteal tendinopathy.

Gluteal tendinopathy (GT) is common and can be debilitating and challenging to manage. A lack of condition specific and appropriate outcome measures compromise evidence synthesis for treatment and limits clinical guideline development. Our objective was to develop a core outcome measurement set for GT (COS-GT). Participants were patients with GT and expert health professionals (HPs). A scoping review identified measures used in GT research, which were mapped to the nine International Scientific Tendinopathy Symposium Consensus core domains, and included in two surveys of HPs. The first survey identified the feasible and true measures for each domain. The second survey refined the list which a patient focus group then considered. Meeting online, HPs reached consensus (agreement ≥70%) on the most appropriate COS-GT measures. 34 HPs and seven patients were recruited. 57 measures were mapped to the nine core domains. Six measures did not proceed past survey one. Of those that progressed, none had adequate clinimetric properties for a COS-GT. Thus, participants decided on interim measures: the global rating of change, pain at night, time to pain onset with single limb stance, pain with stair walking, pain self-efficacy and hip abduction strength. HP participants additionally recommended that pain over the last week, the European Quality of Life-5 dimensions-5 levels and the Victorian Institute of Sport Assessment-Gluteal be considered in clinical trials, as they currently provide best easures of the relevant tendinopathy domains. In conclusion this interim COS-GT should guide outcome measure selection in clinical practice and future research trials in patients with GT.

Sex-Specific Implant Fixation Can Reduce Revision Rates in Total Hip Arthroplasty: Evidence From the Swiss National Joint Registry.

BACKGROUND: Patient's sex is considered a risk factor for revision following primary total hip arthroplasty (THA), but sex-specific treatment guidelines are lacking. The purpose was to assess sex-specificity of risk factors for periprosthetic femoral fractures (PFFs) and aseptic stem loosening (ASL) in a nationwide register study. METHODS: All uncemented and hybrid THAs for hip osteoarthritis registered in the Swiss National Joint Registry were considered. 86,423 THAs were analyzed. Comparable THA subsets for both sexes were obtained through propensity score matching (1:1). A sex-specific analysis of risk factors for early PFF or ASL was performed using recursive partitioning analyses. RESULTS: In women, PFFs were most significantly associated with uncemented THA fixation (P < .0001) and age (P < .01, threshold: 70.5 years). The ASLs were solely associated with patient age of <65 years (P = .023). In men, PFFs were associated exclusively with an American Society of Anesthesiologists (ASA) score >2 (P = .026). The ASLs were not correlated to any of the potential risk factors analyzed. A mathematical simulation indicated that avoiding uncemented THA fixation in women ≥70.5 years of age decreased the number of revisions within the observational period by 21% in this subset and by 4.9% in the entire patient population. CONCLUSION: Uncemented THA should be avoided in women >70.5 years due to the increased risk of early PFF, while the mode of stem fixation did not influence revision risk in men. A sex-specific regimen for THA fixation has the potential to markedly reduce early THA revision rates.

A metabolic throttle regulates the epigenetic state of rDNA.

The synthesis of ribosomal RNA (rRNA) is carefully tuned to match nutritional conditions. In this issue, Murayama et al. (2008) describe a mechanism that couples the energy status of the cell to heterochromatin formation and silencing of rRNA genes. They show that an altered NAD(+)/NADH ratio in response to glucose starvation regulates the silencing activity of eNoSC, a complex consisting of the NAD(+)-dependent histone deacetylase SIRT1, the histone methyltransferase SUV39H1, and a new protein called nucleomethylin (NML). These results suggest a mechanism that links cell physiology to rDNA silencing, which in turn is a prerequisite for nucleolar integrity and cell survival.

ATM, MacroH2A.1, and SASP: The Checks and Balances of Cellular Senescence.

Oncogene activation is usually not enough to induce cancer, but causes cells to arrest proliferation, alter chromatin structure, and increase protein secretion. In this issue of Molecular Cell, Chen et al. (2015) implicate the histone variant macroH2A.1 in the regulation of senescence.

Designing Cell-Type-Specific Genome-wide Experiments.

Multicellular organisms depend on cell-type-specific division of labor for survival. Specific cell types have their unique developmental program and respond differently to environmental challenges, yet are orchestrated by the same genetic blueprint. A key challenge in biology is thus to understand how genes are expressed in the right place, at the right time, and to the right level. Further, this exquisite control of gene expression is perturbed in many diseases. As a consequence, coordinated physiological responses to the environment are compromised. Recently, innovative tools have been developed that are able to capture genome-wide gene expression using cell-type-specific approaches. These novel techniques allow us to understand gene regulation in vivo with unprecedented resolution and give us mechanistic insights into how multicellular organisms adapt to changing environments. In this article, we discuss the considerations needed when designing your own cell-type-specific experiment from the isolation of your starting material through selecting the appropriate controls and validating the data.

ACF takes the driver's seat.

ISWI family chromatin remodeling enzymes generate regularly spaced nucleosome arrays. In a recent Nature report, Hwang et al. (2014) describe how ACF gauges the length of linker DNA when deciding to accelerate nucleosome sliding or to put on the brakes.

Tibial joint line orientation has no effect on joint awareness after mechanically aligned total knee arthroplasty.

PURPOSE: Joint line orientation (JLO) plays an important role in total knee arthroplasty (TKA), but its influence on patient-reported outcomes (PROs) is unclear. The purpose of this study was to examine JLO impact as measured by the forgotten joint score (FJS-12). The hypothesis was that restoring the joint line (JL) parallel to the floor would influence joint awareness favorably, i.e., allow the patient to forget about the joint in daily living. METHODS: All computer-navigated primary TKAs using a cemented, cruciate-retaining (CR) design implanted between January 2018 and September 2019 were reviewed in this retrospective single-center analysis. Primary endpoints were: clinical [range of motion (ROM)], and patient-reported (FJS-12) and radiographical outcomes [tibia joint line angle (TJLA), hip knee axis (HKA), mechanical medial proximal tibia angle (mMPTA) as well as mechanical lateral distal femoral angle (mLDFA)]. RESULTS: Seventy-six patients (mean age: 70.3 ± 9.7 years, mean BMI: 29.7 ± 5.2 kg/m2) were included. Postoperative ROM averaged 118.7 ± 9.6°. The mean FJS-12 improved from 16.4 ± 15.3 (preoperatively) to 89.4 ± 16.9 (1-year follow-up; p < 0.001). Clinical outcomes and PROs did not correlate with JLO (p = n.s.). Cluster analysis using six measures revealed that a medially opened TJLA was associated with significantly better postoperative FJS-12. CONCLUSION: Tibial JLO was found to have no effect on PROs. Considering the JLO in the coronal plane alone probably has questionable clinical relevance. Lower limb alignment should be assessed in all three planes and correlated with the clinical outcome. LEVEL OF CLINICAL EVIDENCE: Level IV.

Different outcomes after proximal femoral replacement in oncologic and failed revision arthroplasty patients - a retrospective cohort study.

BACKGROUND: Proximal femoral replacement (PFR) is a technically demanding procedure commonly performed to restore extensive, oncological or non-oncological bone defects in a severely debilitated patient collective. Depending on different indications, a varying outcome has been reported. The aim of the study was to assess the functional outcomes and complication rates of PFR with the modular Munich-Luebeck (MML) femoral megaprosthesis (ESKA/Orthodynamics, Luebeck, Germany), and to highlight outcome differences in patients treated for failed revision total hip arthroplasty (THA) or malignant bone disease. METHODS: A retrospective review of patients treated with PFR for failed THA or malignant tumor disease between 2000 and 2012 was performed. Patient satisfaction, functional outcome (VAS, SF-12, MSTS, WOMAC, TESS), complications and failure types (Henderson's failure classification) were assessed. A Kaplan-Meier analysis determined implant survival. RESULTS: Fifty-eight patients (age: 69.9 years, BMI: 26.7 kg/m2, mean follow-up: 66 months) were included. The mean SF-12 (physical / mental) was 37.9 / 48.4. MSTS averaged 68% at final follow-up, while mean WOMAC and TESS scored 37.8 and 59.5. TESS and WOMAC scores demonstrated significantly worse outcomes in the revision group (RG) compared to the tumor group (TG). Overall complication rate was 43.1%, and dislocation was the most common complication (27.6%). Implant survival rates were 83% (RG) and 85% (TG; p = n.s.) at 5 years, while 10-year survival was 57% (RG) and 85% (TG, p < 0.05). CONCLUSIONS: PFR is a salvage procedure for restoration of mechanical integrity and limb preservation after extensive bone loss. Complications rates are considerably high. Functional outcomes and 10-year implant survival rate were worse in the RG compared to the TG. Strict indications and disease-specific patient education are essential in preoperative planning and prognosis.

CENPs and Sweet Nucleosomes Face the FACT.

Chaperones mediate vital interactions between histones and DNA during chromatin assembly and reorganization. Two recent studies reveal novel substrates for the essential and conserved histone chaperone FAcilitates Chromatin Transcription (FACT). Prendergast et al. show that FACT helps deposit important histone-fold proteins on centromeres. Raj et al. find that FACT preferentially binds O-GlcNAcylated nucleosomes, suggesting that FACT may contribute to nutrient-regulated cellular programs.

The Metabolic Impact on Histone Acetylation and Transcription in Ageing.

Loss of cellular homeostasis during aging results in altered tissue functions and leads to a general decline in fitness and, ultimately, death. As animals age, the control of gene expression, which is orchestrated by multiple epigenetic factors, degenerates. In parallel, metabolic activity and mitochondrial protein acetylation levels also change. These two hallmarks of aging are effectively linked through the accumulating evidence that histone acetylation patterns are susceptible to alterations in key metabolites such as acetyl-CoA and NAD(+), allowing chromatin to function as a sensor of cellular metabolism. In this review we discuss experimental data supporting these connections and provide a context for the possible medical and physiological relevance.