Biology subjects

Lawrence, J. B.

Publications and source records attributed to Lawrence, J. B..

5 recordsLinked to original sources

bioRxiv2024

Differences in Alu vs L1-rich chromosome bands underpin architectural reorganization of the inactive-X chromosome and SAHFs

The linear DNA sequence of mammalian chromosomes is organized in large blocks of DNA with similar sequence properties, producing a pattern of dark and light staining bands on mitotic chromosomes. Cytogenetic banding is essentially invariant between people and cell-types and thus may be assumed unrelated to genome regulation. We investigate whether large blocks of Alu-rich R-bands and L1-rich G-bands provide a framework upon which functional genome architecture is built. We examine two models of large-scale chromatin condensation: X-chromosome inactivation and formation of senescence-associated heterochromatin foci (SAHFs). XIST RNA triggers gene silencing but also formation of the condensed Barr Body (BB), thought to reflect cumulative gene silencing. However, we find Alu-rich regions are depleted from the L1-rich BB, supporting it is a dense core but not the entire chromosome. Alu-rich bands are also gene-rich, affirming our earlier findings that genes localize at the outer periphery of the BB. SAHFs similarly form within each territory by coalescence of syntenic L1 regions depleted for highly Alu-rich DNA. Analysis of senescent cell Hi-C data also shows large contiguous blocks of G-band and R-band DNA remodel as a segmental unit. Entire dark-bands gain distal intrachromosomal interactions as L1-rich regions form the SAHF. Most striking is that sharp Alu peaks within R-bands resist these changes in condensation. We further show that Chr19, which is exceptionally Alu rich, fails to form a SAHF. Collective results show regulation of genome architecture corresponding to large blocks of DNA and demonstrate resistance of segments with high Alu to chromosome condensation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=179 HEIGHT=200 SRC="FIGDIR/small/574742v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@5ac4b9org.highwire.dtl.DTLVardef@1298a63org.highwire.dtl.DTLVardef@3c8b01org.highwire.dtl.DTLVardef@c9f5f4_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Chromosome silencing in vitro reveals trisomy 21 causes cell-autonomous deficits in angiogenesis and early dysregulation in Notch signaling

Despite the prevalence and clinical importance of Down syndrome, little is known as to the specific cell pathologies that underlie this multi-system disorder. To understand which cell types and pathways are more directly impacted by trisomy 21, we used an inducible-XIST system to silence the extra chromosome 21 in a panel of patient-derived iPSCs. Transcriptomic analysis showed significant dysregulation of Notch signaling occurring as early as pluripotent stem cells, potentially impacting programming of multiple cell-types. Unbiased analysis from iPSCs revealed prominent dysregulation in two major cell type processes: neurogenesis and angiogenesis. Angiogenesis is important for many systems impacted in Down syndrome but has been understudied; therefore, we focused on investigating whether trisomy 21 impacts endothelial cells. An in vitro assay for microvasculature formation used in a tightly controlled system reveals a novel cellular pathology involving delays in angiogenic response during tube formation. Results demonstrate that this is a cell-autonomous effect of trisomy 21, and transcriptomic analysis of differentiated endothelial cells shows deficits in known angiogenesis regulators. This study reveals a major unknown cell pathology caused by trisomy 21 and highlights the importance of endothelial cell function for Down syndrome comorbidities, with wide reaching implications for development and disease progression.

developmental biology↗

bioRxiv2022

Modeling Down syndrome neurodevelopment with isogenic cerebral organoids

As a model of early fetal brain development in Down syndrome, this study examines cortical organoids generated from isogenic trisomic and disomic iPSC lines. Initially pools of organoids from a trisomic versus disomic line found broad transcriptomic differences and modest differences in cell-type representation, suggesting a potential neurodevelopmental phenotype due to Trisomy 21. To better control for multiple sources of variation, we undertook a very robust study of ~1,200 organoids, using an expanded panel of six isogenic subclones (three disomic and three trisomic). The power of the experimental design was indicated by exceptionally strong detection of the ~1.5-fold difference in most chr21 genes. Despite some variability in secreted A{beta}-40 levels between "identical" cell lines, this Alzheimer-related phenotype was detected as clearly correlated with Trisomy 21. However, the many statistically significant non-chr21 DEGs found in the small experiment fell away in the expanded study design, such that just three non-chr21 DEGs correlated to T21 status. Similarly, differences in cell-type representation of organoids varied somewhat between the six isogenic lines, but did not correlate with T21 status. Overall, our results indicate that even when organoid and batch variability are better controlled, common, subtle differences between isogenic cell lines (even subclones) may obscure, or be confused with, differences due to Trisomy 21. Interestingly, the neurodegenerative increase in A{beta} due to T21 was strong enough to be evident in "fetal" organoids. In contrast, any neurodevelopmental phenotype that may be present in the ~2nd trimester of DS brain development may be more subtle, and within the range of variability in neurodifferentiation potential (unrelated to Trisomy 21) of our isogenic iPSC lines. The potential significance of two non-Chr21 DEGs that results suggest correlate with T21 is discussed.

developmental biology↗

bioRxiv2022

Human XIST RNA acts early to condense architecture which facilitates A-repeat density-dependent initiation of gene silencing

XIST RNA triggers gene silencing chromosome-wide and transforms a euchromatic chromosome into a condensed Barr body. XIST is heavily studied in mouse ES cells, but here an inducible iPSC system allows analysis of initial steps in human chromosome silencing, revealing key points not known in either system. XIST RNA distribution was examined relative to biochemical and transcriptional changes directly within architecture of individual chromosome territories. Within a few hours of induction, XIST transcripts distribute as a large "sparse zone" and a smaller "dense zone", which, importantly, exhibit different effects on chromatin. Very sparse transcripts immediately trigger bright staining for H2AK119ub and CIZ1, a structural matrix protein. In contrast, H3K27me3 enrichment comes hours later and is much more restricted to the smaller dense RNA zone, which enlarges as the chromosome condenses. Importantly, silencing of several genes examined occurred well after architectural condensation, suggesting a possibly separable step. Surprisingly, we show the small A-repeat fragment of XIST can alone silence endogenous genes; however, results indicate this requires high local RNA density for effective histone deacetylation. Results support a concept whereby XIST RNA acts directly to condense the chromosome territory, comprised largely of non-coding DNA, which facilitates a required step to initiate gene silencing by the A-repeat. Hence, compacted architecture is not a consequence of collective gene silencing, but an early step required for chromosome-wide gene silencing.

cell biology↗

bioRxiv2021

ZNF146/OZF and ZNF507 target LINE-1 sequences

Repetitive sequences including transposable elements (TEs) and transposon-derived fragments account for nearly half of the human genome. While transposition-competent TEs must be repressed to maintain genomic stability, mutated and fragmented TEs comprising the bulk of repetitive sequences can also contribute to regulation of host gene expression and broader genome organization. Here we analyzed published ChIP-seq data sets to identify proteins broadly enriched on TEs in the human genome. We show two of the proteins identified, C2H2 zinc finger-containing proteins ZNF146 (also known as OZF) and ZNF507, are targeted to distinct sites within LINE-1 ORF2 at thousands of locations in the genome. ZNF146 binding sites are found at old and young LINE-1 elements. In contrast, ZNF507 preferentially binds at young LINE-1 sequences correlated to sequence changes in LINE-1 elements at ZNF507s binding site. To gain further insight into ZNF146 and ZNF507 function, we disrupt their expression in HEK293 cells using CRISPR/Cas9 and perform RNA sequencing, finding modest gene expression changes in cells where ZNF507 has been disrupted. We further identify a physical interaction between ZNF507 and PRMT5, suggesting ZNF507 may target arginine methylation activity to LINE-1 sequences.

cell biology↗