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Periodic Report Summary 1 - BLOODY SIGNALS (Multidimensional analysis of signaling during normal hematopoietic differentiation and stress-induced regeneration)

Description of the project objectives

The foundation of this proposal relies on two major goals. The first goal is to understand how signal responsive transcription factors can synergize with lineage specific regulators and coordinate hematopoietic differentiation during steady state and stress. Previous work by others and us has clearly shown that both hematopoietic differentiation, but also regeneration of blood populations under stress conditions, depend on transcription factors responsible for lineage specification as well as distinct signalling pathways that translate their actions through different transcription factors. For example, BMP signalling is important for erythroid differentiation and SMAD transcription factors, that mediate this pathway, co-regulate gene expression with the lineage regulators GATA1 and GATA2. Our goal is to understand how this interplay changes during diverse stages of differentiation and regeneration, using erythroid lineage as an exemplary system. We want to identify novel regulators of normal and stress erythropoiesis and underpin the mechanism through which signal responsive factors are recruited to chromatin.
Our second goal is to reveal novel interactions between signalling pathways themselves and, more specifically, inflammatory and developmental pathways. These pathways have an already established role in both steady state and stress hematopoiesis. Profound understanding of how these signal-responsive pathways interact with each other can reveal how we can manipulate them therapeutically.

Work performed since the beginning of the project

AIM1. Identify regulators of stress-induced hematopoiesis.
For this aim, we firstly established that BMP signalling is important for erythroid differentiation by using activators and inhibitors of the pathway and examining normal erythroid differentiation. We continued our experiments with a panel of genome-wide datasets (CHIP-seq, RNA-seq, ATAC-seq) that span all consecutive stages of erythroid differentiation of human blood progenitor cells. In parallel, we performed CHIP-seq analysis of lineage and signalling factors in mouse erythroid cells after phenylhydrizine stress, which induces stress erythropoiesis in the spleen.
AIM2. Define how signal-responsive factors are recruited to chromatin.
From AIM1 we observed that SMAD signalling factors co-localise with lineage regulators at a subset of the genomic regions bound by lineage factors. We hypothesized the differences between the GATA/SMAD and GATA only regions will reveal information about SMAD recruitment to chromatin. We performed extensive motif analysis and H3K27ac CHIP-seq experiments to identify differences between these regions. Finally, we used CRIPSR-Cas9 technology to identify the order of recruitment of lineage and signalling factors to chromatin.
AIM3. Interactions between inflammatory and developmental signalling pathways. We conducted a mini screen in zebrafish, based on different receptors that are known to induce inflammatory signalling. We identified novel pathways that are key regulators of hematopoietic stem cell formation and maintenance. RNA-seq analysis revealed that inflammatory pathways lie above many developmental signals.

Main results achieved

AIM1. We have identified the exact genomic regions that can act as hotspots for signalling and lineage regulators during both normal but also stress erythropoiesis. We determined that genes co-bound by signal responsive and lineage regulators exhibit higher expression and change significantly during differentiation. These co-bound regions are localised in super-enhancers and span regions of open chromatin. In contrast, regions that lineage regulators bind to without signalling factors, map into compacted chromatin and play a minimal role in gene expression. During normal differentiation, the binding of lineage regulators is relatively stable between consecutive stages, but during regeneration lineage factors’ binding varies significantly. We have now created a genome wide map of genomic regions bound by both signal responsive and lineage regulators. We consider these regions as genomic hotspots with essential roles in erythropoiesis.
AIM2. By extensive motif analysis during erythroid differentiation we observed that SMAD/GATA regions are surrounded by other major lineage regulators, like Pu.1 in progenitor cells or KLF1 in erythroid cells. However, in regions where lineage regulators bind alone, only generic transcription factor-binding sites could be found. This hints at the fact that lineage regulators may create a hub for signalling factors, thereby permitting them to access these parts of the genome. Upon mutagenising several motifs on the same genomic regions, we observed that knocking out any putative motif from either a lineage regulator or a signal-responsive factor was enough to majorly affect the expression of the neighbouring gene. These results hint at the fact that hotspots’ architecture is important for maintaining gene expression.
AIM3. We have identified a family of inflammatory signalling mediators that play a novel role in hematopoietic stem cell development in zebrafish, but also in adult hematopoietic stem cell maintenance. We have constructed a network of inflammatory signalling factors and determined how they interact with developmental signalling pathways.

Expected final results and their potential impact

We are expecting that, by the end of this grant proposal, we will have achieved the following results: 1) Gain a comparative knowledge in how signal responsive and lineage transcription factors modulate their behaviour during normal or stress erythropoiesis. 2) Understand why and how signalling factors are recruited to certain genomic regions. 3) Create a list of novel regulators of normal and stress erythropoiesis and test some of them functionally. 4) Identify mechanisms by which inflammatory signalling interacts with developmental pathways and regulates hematopoiesis.
By identifying the factors that participate in regulating transcription locally around blood-specific genes during differentiation and regeneration, we should be able to manipulate the process for the benefit of treating blood diseases and for improving marrow and cord blood transplantation.

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Life Sciences
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