Biology subjects

Bothe, M.

Publications and source records attributed to Bothe, M..

7 recordsLinked to original sources

bioRxiv2026

Transcription-dependent heterochromatin at the Xist promoter shapes the random choice of the inactive X chromosome

In female mammals, Xist, the master regulator of X-chromosome inactivation (XCI), is expressed monoallelically. This pattern is established during early embryonic development, when the active Xist allele is chosen at random in each cell. How this choice is made remains incompletely understood. Combining knock-down and overexpression strategies in differentiating mouse embryonic stem cells, which recapitulate the onset of random XCI, we identify a role for the repressive chromatin mark H3K9me3 in the XCI initiation. We show that H3K9me3 accumulates at the promoter-proximal region of the silent Xist allele in female cells as monoallelic expression is established. Unexpectedly, this accumulation requires prior transcription of Xist itself, likely during the initial phase of upregulation, when Xist is frequently transcribed in male cells and from both X chromosomes in females. A repressive function of Xist-dependent H3K9me3 accumulation is supported by our finding that premature, transient Xist overexpression primes an allele for future silencing and skews the choice of the inactive X. Xist-dependent H3K9me3 recruitment does not require its antisense transcript Tsix, which can nonetheless enhance subsequent maintenance of the mark. In addition, the X-linked Xist activator RNF12 counteracts H3K9me3 formation, independently of its known target REX1. Our results thus point to facultative heterochromatin formation as a key contributor to choice at the onset of XCI, where activating and repressing mechanisms are intertwined to establish monoallelic Xist expression.

genetics↗

bioRxiv2024

Reporter CRISPR screens decipher cis- and trans-regulatory principles at the Xist locus

Developmental genes are controlled by an ensemble of cis-acting regulatory elements (REs), which in turn respond to multiple trans-acting transcription factors (TFs). Understanding how a cis-regulatory landscape integrates information from many dynamically expressed TFs has remained a challenge. We develop a combined CRISPR-screening approach using endogenous RNA and RE-reporters as readouts. Applied to the Xist locus, crucial for X-chromosome inactivation in females, this method allows us to comprehensively identify Xist-controlling TFs and map their TF-RE wiring. We find a group of transiently expressed TFs that regulate proximal REs, driving the binary activation of Xist expression. These basal activators are more highly expressed in cells with two X chromosomes, potentially driving female-specific Xist upregulation. A second set of developmental TFs is upregulated later during differentiation and targets distal REs. This regulatory axis is crucial to achieve high levels of Xist RNA, which is necessary for X-chromosome inactivation. Our findings support a model for developmental gene regulation in which factors targeting proximal REs drive binary ON-OFF decisions, while factors interacting with distal REs control the transcription output.

genetics↗

bioRxiv2024

Antisense transcription can induce expression memory via stable promoter repression

The capacity of cells to retain a memory of previous signals enables them to adopt unique cell fates and adjust to their surrounding environment. The underlying gene expression memory can arise from mutual repression of two genes, forming a toggle switch. Such mutual repression may occur at antisense loci, where two convergently oriented genes repress each other in cis. Under which conditions antisense transcription can generate expression memory remains poorly understood. To address this question, we combine mathematical modeling, genomics and a synthetic biology approach. Through simulations we show that stable memory can emerge, if both genes in an antisense pair transcribe through the convergent promoter and induce a stable repressive chromatin state. Genome-wide analysis of nascent transcription further supports antisense-mediated promoter repression with promoter-overlapping antisense gene pairs exhibiting mutually exclusive expression. Through constructing a synthetic antisense locus in mouse embryonic stem cells (mESCs) we then show that such a locus architecture can indeed maintain a memory of a transient stimulus. Mutual repression and the capacity for memory formation are elevated, when mESCs differentiate, showing that epigenetic memory is a cell type-specific property. Our finding that stem cells adapt their ability to remember stimuli as they differentiate might help to elucidate how stemness is maintained.

genetics↗

bioRxiv2021

Glucocorticoid receptor activation induces gene-specific transcriptional memory and universally reversible changes in chromatin accessibility

Glucocorticoids are stress hormones that elicit cellular responses by binding to the glucocorticoid receptor (GR), a ligand-activated transcription factor. The exposure of cells to this hormone induces wide-spread changes in the chromatin landscape and gene expression. Previous studies have suggested that some of these changes are reversible whereas others persist even when the hormone is no longer around. However, when we examined chromatin accessibility in human airway epithelial cells after hormone washout, we found that the hormone-induced changes were universally reversed after one day. Reversibility of hormone-induced changes are found for GR-occupied opening sites and also for closing sites that typically lack GR occupancy. These closing sites are enriched near repressed genes, suggesting that transcriptional repression by GR does not require nearby GR binding. Mirroring what we say in terms of chromatin accessibility, we found that transcriptional responses to hormone are universally reversable. Moreover, priming of cells by a previous exposure to hormone, in general, did not alter the transcriptional response to a subsequent encounter of the same cue. Interestingly, despite the short-lived nature of hormone-induced changes in the chromatin landscape, we identified a single gene, ZBTB16, that displays transcriptional memory manifesting itself as a more robust transcriptional response upon repeated hormone stimulation. Single-cell analysis revealed that the more robust response is driven by a higher probability of primed cells to activate ZBTB16 and by a subset of cells that express the gene at levels that are higher than the induction levels observed for naive cells. Although our study shows that hormone-induced changes are typically reversable, exposure to hormone can induce gene-specific changes in the response to subsequent exposures which may play a role in habituation to stressors and changes in glucocorticoid sensitivity.

genomics↗

bioRxiv2020

Androgen and glucocorticoid receptor direct distinct transcriptional programs by receptor-specific and shared DNA binding sites

The glucocorticoid (GR) and androgen (AR) receptors execute unique functions in vivo, yet have nearly identical DNA binding specificities. To identify mechanisms that facilitate functional diversification among these transcription factor paralogs, we studied AR and GR in an equivalent cellular context. Analysis of chromatin and sequence features suggest that divergent binding, and corresponding gene regulation, are driven by different abilities of AR and GR to interact with relatively inaccessible chromatin. Divergent genomic binding patterns can also be the results of subtle differences in DNA binding preference between AR and GR. Furthermore, the sequence composition of large regions (>10 kb) surrounding selectively occupied binding sites differs significantly, indicating a role for the sequence environment in selectively guiding AR and GR to distinct binding sites. The comparison of binding sites that are shared between AR and GR shows that the specificity paradox can also be resolved by differences in the events that occur downstream of receptor binding. Specifically, we find that shared binding sites display receptor-specific enhancer activity, cofactor recruitment and changes in histone modifications. Genomic deletion of shared binding sites demonstrates their contribution to directing receptor-specific gene regulation. Together, these data suggest that differences in genomic occupancy as well as divergence in the events that occur downstream of receptor binding direct functional diversification among transcription factor paralogs.

genomics↗

bioRxiv2018

Expanding the repertoire of glucocorticoid receptor target genes by engineering genomic response elements

The glucocorticoid receptor (GR), a hormone-activated transcription factor, binds to a myriad of genomic binding sites yet seems to regulate a much smaller number of genes. Genome-wide analysis of GR binding and gene regulation has shown that the likelihood of GR-dependent regulation increases with decreased distance of its binding to the transcriptional start site of a gene. To test if we can adopt this knowledge to expand the repertoire of GR target genes, we used homology directed repair (HDR)-mediated genome editing to add a single GR binding site directly upstream of the transcriptional start site of four genes. To our surprise, we found that the addition of a single GR binding site can be enough to convert a gene into a GR target. The gain of GR-dependent regulation was observed for two out of four genes analyzed and coincided with acquired GR binding at the introduced binding site. However, the gene-specific gain of GR-dependent regulation could not be explained by obvious differences in chromatin accessibility between converted genes and their non-converted counterparts. Further, by introducing GR binding sequences with different nucleotide compositions, we show that activation can be facilitated by distinct sequences without obvious differences in activity between the GR binding sequence variants we tested. The approach to use genome engineering to build genomic response elements facilitates the generation of cell lines with tailored repertoires of GR-responsive genes and a framework to test and refine our understanding of the cis-regulatory logic of gene regulation by testing if engineered response elements behave as predicted.

bioengineering↗

bioRxiv2018

Synthetic STARR-seq reveals how DNA shape and sequence modulate transcriptional output and noise.

The binding of transcription factors to short recognition sequences plays a pivotal role in controlling the expression of genes. The sequence and shape characteristics of binding sites influence DNA binding specificity and have also been implicated in modulating the activity of transcription factors downstream of binding. To quantitatively assess the transcriptional activity of dozens of thousands of designed synthetic sites in parallel, we developed a synthetic version of STARR-seq (synSTARR-seq). We used the approach to systematically analyze how variations in the recognition sequence of the glucocorticoid receptor (GR) affect transcriptional regulation. Our approach resulted in the identification of a novel highly active functional GR binding sequence and revealed that sequence variation both within and flanking GRs core binding site can modulate GR activity without apparent changes in DNA binding affinity. Notably, we found that the sequence composition of variants with similar activity profiles was highly diverse. In contrast, groups of variants with similar activity profiles showed distinct DNA shape characteristics indicating that DNA shape may be a better predictor of activity than DNA sequence. Finally, using single cell experiments with individual enhancer variants, we obtained clues indicating that the architecture of the response element can independently tune expression mean and cell-to cell variability in gene expression (noise). Together, our studies establish synSTARR as a powerful method to systematically study how DNA sequence and shape modulate transcriptional output and noise.

systems biology↗