Loss of the Hematopoietic Stem Cell Factor GATA2 in the Osteogenic Lineage Impairs Trabecularization and Mechanical Strength of Bone

The transcription factor GATA2 is required for expansion and differentiation of hematopoietic stem cells (HSCs). In mesenchymal stem cells (MSCs), GATA2 blocks adipogenesis, but its biological relevance and underlying genomic events are unknown. We report a dual function of GATA2 in bone homeostasis. GATA2 in MSCs binds near genes involved in skeletal system development and colocalizes with motifs for FOX and HOX transcription factors, known regulators of skeletal development. Ectopic GATA2 blocks osteoblastogenesis by interfering with SMAD1/5/8 activation. MSC-specific deletion of GATA2 in mice increases the numbers and differentiation capacity of bone-derived precursors, resulting in elevated bone formation. Surprisingly, MSC-specific GATA2 deficiency impairs the trabecularization and mechanical strength of bone, involving reduced MSC expression of the osteoclast inhibitor osteoprotegerin and increased osteoclast numbers. Thus, GATA2 affects bone turnover via MSC-autonomous and indirect effects. By regulating bone trabecularization, GATA2 expression in the osteogenic lineage may contribute to the anatomical and cellular microenvironment of the HSC niche required for hematopoiesis.Peer reviewe

An Analysis of Transcriptional Memory and Mechanisms That Direct Divergent Genomic Occupancy of Related Transcription Factors

The glucocorticoid receptor (GR) is a transcription factor which becomes activated upon binding to glucocorticoids, a class of steroid hormones. Upon activation, GR binds to various genomic locations and induces large-scale changes in transcription and chromatin structure. Clinically, GR is an important therapeutic target, since glucocorticoids are widely applied to treat autoimmune and inflammatory conditions. However, long-term treatment with glucocorticoids is associated with glucocorticoid-resistance and severe side-effects. At the molecular level, the effects of prolonged GR activation on a cell’s transcriptional responses are not fully understood. Here, I investigated if exposure to glucocorticoids results in long-term changes in chromatin and transcription. In addition, I studied GR’s genomic binding preferences by investigating mechanisms that shape DNA binding specificities between GR and its paralog, the androgen receptor (AR). In the first part of the thesis, I investigated the immediate and the long-term effects of GR activation on chromatin and transcription. By examining GR binding as well as glucocorticoid-induced changes in chromatin accessibility and transcription, I found that genomic regions that lose chromatin accessibility were enriched near downregulated genes. Interestingly, these ‘closing’ regions were largely not bound by GR, indicating that repression, in part, does not depend on nearby GR binding and might occur through indirect effects of GR activation. To study the long-term effects of GR activation, I investigated changes in chromatin accessibility and transcription after washout of glucocorticoids. GR-induced changes in chromatin accessibility were found to be reversible following a 24-hour washout period. Similarly, transcriptional activity reverted to basal levels after washout. Moreover, I tested if a prior exposure to hormone changes the response to a subsequent treatment. Most genes showed similar transcriptional responses upon hormone-re-stimulation compared to the first stimulation. However, the GR-target gene ZBTB16 showed enhanced upregulation upon reinduction, suggesting that prior glucocorticoid exposure results in priming of this gene. Single-cell analysis showed that enhanced expression of ZBTB16 upon reinduction was a consequence of an increased probability of cells transcribing the gene as well as individual cells showing increased ZBTB16 transcription. In the second part of the thesis, I assayed the role of chromatin and DNA sequence in generating divergent genomic binding patterns of transcription factors with nearly identical DNA-binding preferences, specifically, GR and its paralog AR. Investigating binding of GR and AR in the same cell type revealed that both transcription factors occupy overlapping as well as unique sites. Examining the chromatin landscape at receptor binding regions showed that many GR-specific sites were situated within relatively inaccessible chromatin, suggesting that binding specificity is, in part, achieved through GR’s ability to bind to inaccessible chromatin. Furthermore, motif enrichment analysis at GR- and AR-specific regions provided further evidence that the receptors exhibit subtle differences in the recognition sequences they preferentially bind to. Lastly, analysis of GC-content revealed that receptor-specific binding is also driven by GC-content at the binding sites and the larger surrounding area, as mean GC-content was found to to be higher at GR- compared to AR-specific sites. In summary, these results provide evidence that GR is capable of inducing gene-specific transcriptional memory, even though GR-induced chromatin structural and transcriptional changes are largely reversible. Given GR’s biological role as an effector to fluctuating levels of glucocorticoids, the reversibility of GR-induced chromatin and transcriptional changes is to be expected. However, future experiments involving longer, or more frequently repeated, hormone exposures might yield insights into the underlying mechanisms of glucocorticoid resistance and side-effects associated with long-term glucocorticoid treatment. Furthermore, the chromatin landscape as well as DNA sequence composition contribute to driving receptor-specific genomic occupancy of GR and AR. These findings might represent a general mechanism that shapes differential binding among paralogous transcription factors and could contribute to our understanding of how genomic binding specificities are established for other related transcription factors.Der Glukokortikoidrezeptor (GR) ist ein Transkriptionsfaktor (TF), der durch Bindung an Glukokortikoide, die zu den Steroidhormonen gehören, aktiviert wird. Aktivierter GR bindet zahlreiche genomische Regionen und induziert weitreichende Veränderungen der Transkription und Chromatin-Struktur. Aus klinischer Sicht stellt GR ein wichtiges Medikamentenziel dar, da Glukokortikoide oft zur Behandlung von Autoimmunkranheiten und Entzündungszuständen eingesetzt werden. Jedoch kann eine Langzeitbehandlung mit Glukokortikoiden zu schwerwiegenden Nebenwirkungen und Glukokortikoid-Resistenzen führen. Inwiefern sich eine Langzeitaktivierung von GR auf molekularer Ebene auswirkt, ist noch nicht klar. In meiner Doktorarbeit habe ich mich mit den Langzeitfolgen nach GR-Aktivierung befasst, die sich auf Chromatin-Struktur und Transkription auswirken. Zusätzlich habe ich die genomischen Bindungspräferenzen von GR untersucht und diese mit den Präferenzen des verwandten Androgenrezeptors (AR) verglichen. Im ersten Teil meiner Doktorarbeit befasste ich mich mit den kurz- und längerfristigen Auswirkungen auf Transkription und Chromatin nach einer GR-Aktivierung. Zu diesem Zweck untersuchte ich genomweite GR-Bindestellen, Veränderungen in der Chromatin-Zugänglichkeit genomischer Regionen und transkriptionelle Veränderungen. Ich konnte zeigen, dass genomische Regionen, die an Chromatin-Zugänglichkeit verlieren und nicht von GR gebunden sind, in der Nähe von reprimierten Genen angereichert waren. Diese Erkenntnisse legen nahe, dass Glukokortikoid-induzierte transkriptionelle Repression mancher Gene ohne lokaler Binding von GR stattfinden kann. Darüber hinaus untersuchte ich, ob Glukokortikoid-induzierte Veränderungen der Chromatin-Zugänglichkeit, über einen längeren Zeitraum bestehen bleiben können oder ob sie reversibel sind. Ich fand, dass die erhöhte oder verringerte Chromatin-Zugänglichkeit infolge einer einmaligen GR-Aktivierung 24 Stunden nach dem Auswaschen der Glukokortikoide reversibel war. Ähnlich verhielt sich die transkriptionelle Aktivität, welche nach dem Auswaschen von Glukokortikoiden wieder auf Basallevel zurückfiel. Außerdem untersuchte ich den Einfluss einer vorherigen Hormon-Exposition auf Transkription nach einer zweiten Stimulation. Die meisten Gene zeigten eine ähnliche transkriptionelle Antwort nach einer solchen Re-stimulation im Vergleich zur ersten Hormonexposition. Jedoch zeigte das GR-Zielgen ZBTB16 eine gesteigerte Hochregulierung nach Re-induktion, was eine Sensibilisierung dieses Gens durch eine vorherige Exposition nahelegt. Einzelzellanalyse nach Re-induktion konnte zeigen, dass die gesteigerte Expression von ZBTB16 eine Konsequenz der erhöhten Wahrscheinlichkeit von Genexpression sowie höherer Expressionsraten einzelner Zellen ist. Im zweiten Teil der Arbeit fokussierte ich mich auf die Rolle von Chromatin und DNA-Sequenz in der Etablierung von spezifischen Bindeverhalten von TF mit fast identischen DNA-Bindepräferenzen am Beispiel von GR und AR. Die Analyse von GR- und AR-Bindepräferenzen im gleichen zellulären Hintergrund konnte zeigen, dass beide TF überlappende sowie spezifische Bindestellen targetieren. Die Untersuchung der Chromatinlandschaft von Rezeptorbindestellen zeigte, dass viele GR spezifische Bindestellen in relativ unzugänglichem Chromatin liegen, was suggeriert, dass GR Spezifität über die Fähigkeit geschlossenes Chromatin zu binden vermittelt wird. Außerdem konnten Sequenzmotifanalysen von GR- und AR-spezifischer Binderegionen weiter auf subtile Differenzen in Erkennungssequenzen hinweisen. Abschließend zeigte eine GC-Gehaltsanalyse, dass rezeptorspezifisches Binden auch vom GC-Gehalt der Bindestellen sowie der weiteren Umgebung abhängt, da der durchschnittliche GC-Gehalt höher an GR- gegenüber AR-spezifischen Bindestellen war. Zusammenfassend konnten diese Ergebnisse zeigen, dass GR genspezifische transkriptionelle Erinnerung induzieren kann, obwohl GR-induzierte Änderungen in Chromatin-Struktur und Transkription größtenteils reversibel sind. Da GR als Effektor fluktuierender Glukokortikoid-Spiegel, aufgrund natürlicher Tagesschwankungen, fungiert, ist eine Reversibilität der GR-induzierten Änderungen teilweise zu erwarten. Zukünftige Experimente könnten längere oder häufiger wiederholte Hormon-Behandlungen beinhalten, um die molekularen Mechanismen, welche den Nebenwirkungen und Glukokortikoid-Resistenzen einer Langzeitbehandlung mit Glukokortikoiden zugrunde liegen, vollständig zu ergründen. Zusätzlich tragen Chromatin-Landschaft sowie DNA-Sequenz zum rezeptorspezifischen genomischen Bindeverhaltens von GR und AR bei. Diese Erkenntnisse könnten möglicherweise generelle Mechanismen spezifischen Verhaltens paraloger TF darstellen und deshalb zu unserem Verständnis der genomischen Bindepräferenzen anderer verwandter TF beitragen

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

International audienceThe 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 tens 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 GR's 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 specific 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

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 them in an equivalent cellular context. Analysis of chromatin and sequence 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 result 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 guiding AR and GR to distinct binding sites. The comparison of binding sites that are shared shows that the specificity paradox can also be resolved by differences in the events that occur downstream of receptor binding. Specifically, 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