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1. Enhancers with cooperative Notch binding sites are more resistant to regulation by the Hairless co-repressor

   https://doi.org/10.1371/journal.pgen.1009039  

   Kuang, Yi; Pyo, Anna; Eafergan, Natanel; Cain, Brittany; Gutzwiller, Lisa M.; Axelrod, Ofri; Gagliani, Ellen K.; Weirauch, Matthew T.; Kopan, Raphael; Kovall, Rhett A.; et al (September 2021, PLOS Genetics)

   Aster, Jon Christopher (Ed.)

   Notch signaling controls many developmental processes by regulating gene expression. Notch-dependent enhancers recruit activation complexes consisting of the Notch intracellular domain, the C bf/ S u(H)/ L ag1 (CSL) transcription factor (TF), and the Mastermind co-factor via two types of DNA sites: monomeric CSL sites and cooperative dimer sites called S u(H) p aired s ites (SPS). Intriguingly, the CSL TF can also bind co-repressors to negatively regulate transcription via these same sites. Here, we tested how synthetic enhancers with monomeric CSL sites versus dimeric SPSs bind Drosophila Su(H) complexes in vitro and mediate transcriptional outcomes in vivo . Our findings reveal that while the Su(H)/Hairless co-repressor complex similarly binds SPS and CSL sites in an additive manner, the Notch activation complex binds SPSs, but not CSL sites, in a cooperative manner. Moreover, transgenic reporters with SPSs mediate stronger, more consistent transcription and are more resistant to increased Hairless co-repressor expression compared to reporters with the same number of CSL sites. These findings support a model in which SPS containing enhancers preferentially recruit cooperative Notch activation complexes over Hairless repression complexes to ensure consistent target gene activation. 

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2. Filamin C Cardiomyopathy Variants Cause Protein and Lysosome Accumulation

   https://doi.org/10.1161/CIRCRESAHA.120.317076  

   Agarwal, Radhika; Paulo, Joao A.; Toepfer, Christopher N.; Ewoldt, Jourdan K.; Sundaram, Subramanian; Chopra, Anant; Zhang, Qi; Gorham, Joshua; DePalma, Steven R.; Chen, Christopher S.; et al (September 2021, Circulation Research)

   Rationale: Dominant heterozygous variants in filamin C ( FLNC ) cause diverse cardiomyopathies, although the underlying molecular mechanisms remain poorly understood. Objective: We aimed to define the molecular mechanisms by which FLNC variants altered human cardiomyocyte gene and protein expression, sarcomere structure, and contractile performance. Methods and Results: Using CRISPR/Cas9, we introduced FLNC variants into human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs). We compared isogenic hiPSC-CMs with normal (wild-type), ablated expression ( FLNC −/− ), or haploinsufficiency ( FLNC +/− ) that causes dilated cardiomyopathy. We also studied a heterozygous in-frame deletion ( FLNC +/Δ7aa ) which did not affect FLNC expression but caused aggregate formation, similar to FLNC variants associated with hypertrophic cardiomyopathy. FLNC −/− hiPSC-CMs demonstrated profound sarcomere misassembly and reduced contractility. Although sarcomere formation and function were unaffected in FLNC +/ − and FLNC +/Δ7aa hiPSC-CMs, these heterozygous variants caused increases in lysosome content, enhancement of autophagic flux, and accumulation of FLNC-binding partners and Z-disc proteins. Conclusions: FLNC expression is required for sarcomere organization and physiological function. Variants that produce misfolded FLNC proteins cause the accumulation of FLNC and FLNC-binding partners which leads to increased lysosome expression and activation of autophagic pathways. Surprisingly, similar pathways were activated in FLNC haploinsufficient hiPSC-CMs, likely initiated by the loss of stoichiometric FLNC protein interactions and impaired turnover of proteins at the Z-disc. These results indicate that both FLNC haploinsufficient variants and variants that produce misfolded FLNC protein cause disease by similar proteotoxic mechanisms and indicate the therapeutic potential for augmenting protein degradative pathways to treat a wide range of FLNC -related cardiomyopathies. 

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3. Affinity-optimizing enhancer variants disrupt development

   https://doi.org/10.1038/s41586-023-06922-8  

   Lim, Fabian; Solvason, Joe J.; Ryan, Genevieve E.; Le, Sophia H.; Jindal, Granton A.; Steffen, Paige; Jandu, Simran K.; Farley, Emma K. (February 2024, Nature)

   Abstract Enhancers control the location and timing of gene expression and contain the majority of variants associated with disease^1–3. The ZRS is arguably the most well-studied vertebrate enhancer and mediates the expression ofShhin the developing limb⁴. Thirty-one human single-nucleotide variants (SNVs) within the ZRS are associated with polydactyly^4–6. However, how this enhancer encodes tissue-specific activity, and the mechanisms by which SNVs alter the number of digits, are poorly understood. Here we show that the ETS sites within the ZRS are low affinity, and identify a functional ETS site, ETS-A, with extremely low affinity. Two human SNVs and a synthetic variant optimize the binding affinity of ETS-A subtly from 15% to around 25% relative to the strongest ETS binding sequence, and cause polydactyly with the same penetrance and severity. A greater increase in affinity results in phenotypes that are more penetrant and more severe. Affinity-optimizing SNVs in other ETS sites in the ZRS, as well as in ETS, interferon regulatory factor (IRF), HOX and activator protein 1 (AP-1) sites within a wide variety of enhancers, cause gain-of-function gene expression. The prevalence of binding sites with suboptimal affinity in enhancers creates a vulnerability in genomes whereby SNVs that optimize affinity, even slightly, can be pathogenic. Searching for affinity-optimizing SNVs in genomes could provide a mechanistic approach to identify causal variants that underlie enhanceropathies. 

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4. The Drosophila HP1 family is associated with active gene expression across chromatin contexts

   https://doi.org/10.1093/genetics/iyab108  

   Schoelz, John M; Feng, Justina X; Riddle, Nicole C (July 2021, Genetics)

   Kaplan, C D (Ed.)

   Abstract Drosophila Heterochromatin Protein 1a (HP1a) is essential for heterochromatin formation and is involved in transcriptional silencing. However, certain loci require HP1a to be transcribed. One model posits that HP1a acts as a transcriptional silencer within euchromatin while acting as an activator within heterochromatin. However, HP1a has been observed as an activator of a set of euchromatic genes. Therefore, it is not clear whether, or how, chromatin context informs the function of HP1 proteins. To understand the role of HP1 proteins in transcription, we examined the genome-wide binding profile of HP1a as well as two other Drosophila HP1 family members, HP1B and HP1C, to determine whether coordinated binding of these proteins is associated with specific transcriptional outcomes. We found that HP1 proteins share many of their endogenous binding targets. These genes are marked by active histone modifications and are expressed at higher levels than nontarget genes in both heterochromatin and euchromatin. In addition, HP1 binding targets displayed increased RNA polymerase pausing compared with nontarget genes. Specifically, colocalization of HP1B and HP1C was associated with the highest levels of polymerase pausing and gene expression. Analysis of HP1 null mutants suggests these proteins coordinate activity at transcription start sites to regulate transcription. Depletion of HP1B or HP1C alters expression of protein-coding genes bound by HP1 family members. Our data broaden understanding of the mechanism of transcriptional activation by HP1a and highlight the need to consider particular protein–protein interactions, rather than broader chromatin context, to predict impacts of HP1 at transcription start sites. 

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5. Germline BARD1 variants predispose to mesothelioma by impairing DNA repair and calcium signaling

   https://doi.org/10.1073/pnas.2405231121  

   Novelli, Flavia; Yoshikawa, Yoshie; Vitto, Veronica_Angela Maria; Modesti, Lorenzo; Minaai, Michael; Pastorino, Sandra; Emi, Mitsuru; Kim, Jin-Hee; Kricek, Franz; Bai, Fang; et al (July 2024, Proceedings of the National Academy of Sciences)

   We report that ~1.8% of all mesothelioma patients and 4.9% of those younger than 55, carry rare germline variants of the BRCA1 associated RING domain 1 (BARD1)gene that were predicted to be damaging by computational analyses. We conducted functional assays, essential for accurate interpretation of missense variants, in primary fibroblasts that we established in tissue culture from a patient carrying the heterozygousBARD1^V523Amutation. We found that these cells had genomic instability, reduced DNA repair, and impaired apoptosis. Investigating the underlying signaling pathways, we found that BARD1 forms a trimeric protein complex with p53 and SERCA2 that regulates calcium signaling and apoptosis. We validated these findings inBARD1-silenced primary human mesothelial cells exposed to asbestos. Our study elucidated mechanisms ofBARD1activity and revealed that heterozygous germlineBARD1mutations favor the development of mesothelioma and increase the susceptibility to asbestos carcinogenesis. These mesotheliomas are significantly less aggressive compared to mesotheliomas in asbestos workers. 

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