Biology subjects

Tawil, R.

Publications and source records attributed to Tawil, R..

8 recordsLinked to original sources

bioRxiv2024

SMCHD1 maintains heterochromatin and genome compartments in human myoblasts

Mammalian genomes are subdivided into euchromatic A compartments that contain mostly active chromatin, and inactive, heterochromatic B compartments. However, it is unknown how A and B genome compartments are established and maintained. Here we studied SMCHD1, an SMC-like protein in human male myoblasts. SMCHD1 colocalizes with Lamin B1 and the heterochromatin mark H3K9me3. Loss of SMCHD1 leads to extensive heterochromatin depletion at the nuclear lamina and acquisition of active chromatin states along all chromosomes. In absence of SMCHD1, long range intra-chromosomal and inter-chromosomal contacts between B compartments are lost while many new TADs and loops are formed. Inactivation of SMCHD1 promotes numerous B to A compartment transitions accompanied by activation of silenced genes. SMCHD1 functions as an anchor for heterochromatin domains ensuring that these domains are inaccessible to epigenome modification enzymes that typically operate in active chromatin. Therefore, A compartments are formed by default when not prevented by SMCHD1.

genomics↗

bioRxiv2023

Validation of the association between MRI and gene signatures in facioscapulohumeral dystrophy muscle: implications for clinical trial design

Identifying the aberrant expression of DUX4 in skeletal muscle as the cause of facioscapulohumeral dystrophy (FSHD) has led to rational therapeutic development and clinical trials. Several studies support the use of MRI characteristics and the expression of DUX4-regulated genes in muscle biopsies as biomarkers of FSHD disease activity and progression, but reproducibility across studies needs further validation. We performed lower-extremity MRI and muscle biopsies in the mid-portion of the tibialis anterior (TA) muscles bilaterally in FSHD subjects and validated our prior reports of the strong association between MRI characteristics and expression of genes regulated by DUX4 and other gene categories associated with FSHD disease activity. We further show that measurements of normalized fat content in the entire TA muscle strongly predict molecular signatures in the mid-portion of the TA. Together with moderate-to-strong correlations of gene signatures and MRI characteristics between the TA muscles bilaterally, these results suggest a whole muscle model of disease progression and provide a strong basis for inclusion of MRI and molecular biomarkers in clinical trial design.

molecular biology↗

bioRxiv2022

Engineered SMCHD1 and D4Z4 mutations reveal roles of D4Z4 heterochromatin disruption and feedforward DUX4 network activation in FSHD

Facioscapulohumeral dystrophy (FSHD) is commonly associated with contraction of D4Z4 repeats on chromosome 4q (FSHD1). Mutations in the SMCHD1 gene are linked to both minor cases with no prominent repeat loss (FSHD2) and severe cases of FSHD1. Abnormal upregulation of the transcription factor DUX4, encoded in the D4Z4 repeat, is believed to play a central role in FSHD. However, defining the disease mechanism has been hampered by the heterogeneity of patient-derived cells, difficulty to detect DUX4 in patient myocytes, and limited animal models because D4Z4 repeats are primate-specific. To overcome these limitations, we engineered isogenic human skeletal myoblast lines with D4Z4 and/or SMCHD1 mutations. We found a highly synergistic effect of double mutations on triggering two key disease processes, D4Z4 heterochromatin disruption and cross-stimulation of DUX4 targets, such as histone H3.X/Y and LEUTX transcription factor. Thus, engineered human myocyte models provide unique insights into the molecular mechanisms underpinning FSHD. TeaserFSHD mutations cause D4Z4 heterochromatin disruption and feedforward DUX4 network activation.

genetics↗

bioRxiv2021

Muscle group specific transcriptomic and DNA methylation differences related to developmental patterning in FSHD

Muscle groups throughout the body are specialized in function and are specified during development by position specific gene regulatory networks. In developed tissue, myopathies affect muscle groups differently. Facioscapulohumeral muscular dystrophy, FSHD, affects upper body and tibialis anterior (TA) muscles earlier and more severely than others such as quadriceps. To investigate an epigenetic basis for susceptibility of certain muscle groups to disease, we perform DNA methylation and RNA sequencing on primary patient derived myoblasts from TA and quadricep for both control and FSHD2 as well as RNA-seq for myoblasts from FSHD1 deltoid, bicep and TA over a time course of differentiation. We find that TA and quadricep retain methylation and expression differences in transcription factors that are key to muscle group specification during embryogenesis. FSHD2 patients have differences in DNA methylation and expression related to SMCHD1 mutations and FGF signaling. Genes induced specifically in FSHD are more highly expressed in commonly affected muscle groups. We find a set of genes that distinguish more susceptible muscle groups including development-associated TFs and genes involved in WNT signaling. Adult muscle groups therefore retain transcriptional and DNA methylation differences associated with development, which may contribute to susceptibility in FSHD.

genomics↗

bioRxiv2020

Relationship of DUX4 and target gene expression in FSHD myocytes

Facioscapulohumeral dystrophy (FSHD) is linked to misexpression of the transcription factor, DUX4. Although DUX4 target gene expression is often readily detectable, analysis of DUX4 expression has been limited due to its low expression in patient samples. Recently, single cell/nucleus RNA-sequencing was used to detect the native expression of DUX4 for the first time, but important spatial relationships with its target gene expression was missing. Furthermore, dynamics of DUX4 expression during myoblast differentiation has not been fully explored. In order to study the spatiotemporal relationship of DUX4 and key target genes, we performed RNA FISH on immortalized FSHD2 patient skeletal muscle cells. Using two probe sets, DUX4 transcripts were detected in 1-4% of myotubes after 3-day differentiation in vitro. We found that DUX4 transcripts mainly localize as foci in one or two nuclei in a myotube compared to abundant accumulation of the target gene transcripts in the cytoplasm. Over a 13-day differentiation timecourse, DUX4 expression without target gene expression significantly increased and peaked at day 7. Target gene expression correlates better with DUX4 expression early in differentiation while the expression of target genes without detectable DUX4 transcripts increases later. Consistently, shRNA depletion of DUX4-activated transcription factors, DUXA and LEUTX, specifically repressed a DUX4-target gene, KDM4E, later in differentiation, suggesting that following the initial activation by DUX4, target genes themselves contribute to the maintenance of downstream gene expression. Together, in situ detection of the DUX4 and target gene transcripts provided new insight into dynamics of DUX4 transcriptional network in FSHD patient myocytes. Significance StatementFSHD is the third most common muscular dystrophy and is associated with upregulation of DUX4, a transcription factor, and its target genes. Although target genes are easily detectable in FSHD, low frequency DUX4 upregulation in patient myocytes is difficult to detect, and examining the relationship and dynamics of DUX4 and target gene expression without artificial overexpression of DUX4 has been challenging. Using RNAScope with highly specific probes, we detect the endogenous DUX4 and target gene transcripts in situ in patient skeletal myotubes during differentiation in vitro. Our study reveals a unique DUX4 expression pattern and its relationship to the expression of target genes, and evidence for self-sustainability of the target gene network. The study provides important new insights into the FSHD disease mechanism.

genetics↗

bioRxiv2019

Cytosolic Proteostatic Adaptation to Mitochondrial Stress Causes Progressive Muscle Wasting

Mitochondrial dysfunction causes muscle wasting (or atrophy) in many diseases and probably also during aging. The underlying mechanism is unclear. Accumulating evidence suggests that substantial levels of bioenergetic deficiency and oxidative stress are insufficient by themselves to intrinsically cause muscle wasting, raising the possibility that non-bioenergetic factors may contribute to mitochondria-induced muscle wasting. In this report, we show that chronic adaptation to mitochondria-induced proteostatic stress in the cytosol induces muscle wasting. We generated transgenic mice with unbalanced mitochondrial protein loading and import, by a two-fold increase in the expression of the nuclear-encoded mitochondrial carrier protein, Ant1. We found that the ANT1-transgenic mice progressively lose muscle mass. Skeletal muscle is severely atrophic in older mice without affecting the overall lifespan. Mechanistically, Ant1 overloading induces aggresome-like structures and the expression of small heat shock proteins in the cytosol. The data support mitochondrial Precursor Overaccumulation Stress (mPOS), a recently discovered cellular stress mechanism caused by the toxic accumulation of unimported mitochondrial precursors/preproteins. Importantly, the ANT1-transgenic muscles have a drastically remodeled transcriptome that appears to be trying to counteract mPOS, by repressing protein synthesis, and by stimulating proteasomal function, autophagy and lysosomal amplification. These anti-mPOS responses collectively reduce protein content, which is known to decrease myofiber size and muscle mass. Our work therefore revealed that a subtle imbalance between mitochondrial protein load and import is sufficient to induce mPOS in vivo, and that anti-mPOS adaptation is a robust mechanism of muscle wasting. This finding may help improve the understanding of how mitochondria contribute to muscle wasting. It could have direct implications for several human diseases associated with ANT1 overexpression, including Facioscapulohumeral Dystrophy (FSHD). One Sentence SummaryProteostatic adaptations to proteostatic stress in the cytosol caused by unbalanced mitochondrial protein loading and import lead to progressive muscle wasting.

cell biology↗

bioRxiv2019

P38α Regulates Expression of DUX4 in Facioscapulohumeral Muscular Dystrophy

FSHD is caused by the loss of repression at the D4Z4 locus leading to DUX4 expression in skeletal muscle, activation of its early embryonic transcriptional program and muscle fiber death. While progress toward understanding the signals driving DUX4 expression has been made, the factors and pathways involved in the transcriptional activation of this gene remain largely unknown. Here, we describe the identification and characterization of p38 as a novel regulator of DUX4 expression in FSHD myotubes. By using multiple highly characterized, potent and specific inhibitors of p38/{beta}, we show a robust reduction of DUX4 expression, activity and cell death across FSHD1 and FSHD2 patient-derived lines. RNA-seq profiling reveals that a small number of genes are differentially expressed upon p38/{beta} inhibition, the vast majority of which are DUX4 target genes. Our results reveal a novel and apparently critical role for p38 in the aberrant activation of DUX4 in FSHD and support the potential of p38/{beta} inhibitors as effective therapeutics to treat FSHD at its root cause. VISUAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=160 SRC="FIGDIR/small/700195v2_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@d2d55corg.highwire.dtl.DTLVardef@1b5c8fcorg.highwire.dtl.DTLVardef@19e4667org.highwire.dtl.DTLVardef@202b96_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2018

Single-nucleus RNA-seq identifies divergent populations of FSHD2 myotube nuclei

FSHD is characterized by the misexpression of DUX4 in skeletal muscle. However, DUX4 is lowly expressed in patient samples and analysis of the consequences of DUX4 expression has largely relied on artificial overexpression. To better understand the native expression profile of DUX4 and its targets, we performed pooled RNA-seq differentiation time-course in FSHD2 patient-derived primary myoblasts and identified early-and late-induced sets of FSHD-associated genes. Using single-cell and single-nucleus RNA-seq on FSHD2 myoblasts and myotubes respectively, we captured DUX4 expression in single-nuclei and found that only some DUX4 targets are coexpressed. We identified two populations of FSHD myotube nuclei with distinct transcriptional profiles. One population is highly enriched with DUX4 and FSHD related genes, including the DUX4 paralog DUXA ("FSHD-Hi"). The other population has no expression of DUX4 and expresses low amounts of FSHD related genes ("FSHD-Lo"), but is marked by the expression of CYTL1 and CHI3L1. "FSHD-Hi" myotube nuclei upregulated a set of transcription factors (TFs) that may form a self-sustaining network of gene dysregulation, which perpetuates this disease after DUX4 is no longer expressed.

genomics↗