Remodel your way to fetal hematopoiesis

In this issue of Blood, the study by Ding et al¹  describes a novel role for the chromatin remodeling factor smarca5 during the development of hematopoietic stem and progenitor cells (HSPCs) as they emerge from dorsal aorta hemogenic endothelium^2-4  and then transition to fetal-like stage.^5,6 

Throughout vertebrate hematopoietic ontogeny there is always a fetal-like stage between HSPC emergence and the adult marrow.⁷  Changes in the genetic program of HSPCs are apparent from the moment they leave the dorsal aorta⁸  and as they transit through the fetal niche. However, how an HSPC undergoes these intrinsic changes is not well understood.

To explore these intriguing events, the authors follow chromatin and transcriptome changes in zebrafish HSPCs. The zebrafish is ideal to study these processes because of its highly conserved hematopoietic system and many available genetic tools. First, the authors used cell sorting to purify labeled HSPCs from transgenic zebrafish lines (cd41:gfp⁺/gata1:dsRed⁻), collecting cells from either nascent or fetal-like stages. In zebrafish, fetal-like hematopoiesis progresses in a vascular region of the embryonic tail called the caudal hematopoietic tissue (CHT),^5,6  which is equivalent to the mammalian fetal liver. Using nascent or fetal-like HSPCs, they performed assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing (RNA-seq) to confirm a difference in chromatin accessibility and gene expression, respectively, between the 2 stages. The analysis revealed there is an overall increase in chromatin accessibility of hematopoietic genes during the nascent to fetal-like transition, suggesting progression in the developmental program.

To find the chromatin remodeling factors that may be responsible for this increase in chromatin accessibility, the authors examined the expression profile of 65 factors between nascent and fetal-like stages and then focused on the factors that were upregulated during the transition. They further narrowed these candidates by using morpholino knockdown, a rapid screening tool in zebrafish for testing gene function. There were 7 chromatin remodeling factors that upon knockdown had fewer HSPCs in the fetal-like CHT but had no effect on HSPC numbers at earlier emergence stages. After considering the spatial and temporal expression pattern of each factor, they decided to further pursue smarca5 because of its increased and specific expression in fetal-like HSPCs.

A smarca5 mutant generated by CRISPR/Cas9 recapitulated the morpholino phenotype and showed similar reduction in definitive HSPCs from fetal-like stages and later. To address their primary hypothesis that smarca5 is necessary for increased chromatin accessibility during the nascent to fetal-like transition of HSPCs, the authors compared ATAC-seq from smarca5 mutant and control HSPCs. Importantly, they found reduced chromatin accessibility in smarca5 mutant HSPCs that occurred primarily around hematopoietic maintenance and differentiation genes.

To further understand the interaction of smarca5 with various regions of chromatin, the zebrafish model was again leveraged by creation of an inducible transgenic line for in vivo expression of FLAG-tagged smarca5 under control of a heat shock promoter (hsp70:FLAG-smarca5-EGFP). Using this approach, the authors could perform chromatin immunoprecipitation sequencing (ChIP-seq) experiments specifically at the fetal-like stage of hematopoiesis after a brief induction of FLAG-tagged-smarca5. One of the key findings from these ChIP-seq experiments was that chromatin regions bound by smarca5 were the same regions that had reduced accessibility in ATAC-seq from smarca5 mutants. These data support a highly specific role for smarca5 in chromatin remodeling during the transition from nascent to fetal-like hematopoiesis.

Next, the authors were able to use the same inducible FLAG-tagged smarca5 transgenic line to discover potential in vivo protein binding partners. Mass spectrometry showed that one of the top candidates for protein-protein interaction was the nucleolar protein nucleolin, which is encoded by the gene ncl. Interestingly, ncl shared the same expression pattern as smarca5 in the fetal-like CHT niche, and a CRISPR/Cas9 ncl mutant had similar defects in fetal-like HSPC development. Creating another inducible FLAG-tagged transgenic line (hsp70:FLAG-ncl-EGFP), the authors could perform in vivo ChIP-seq for nucleolin and found significant overlap with smarca5 binding sites.

The authors established that smarca5 and nucleolin form a chromatin remodeling complex that functions in the transition to fetal-like hematopoiesis (see figure). Therefore, it was important to understand how these factors promote the changing gene regulatory landscape of developing fetal-like HSPCs. The finding that hematopoiesis-related transcription factor binding motifs were enriched in smarca5-bound regions, including klf1 and spi1, started to reveal an underlying gene regulatory network. To identify smarca5 targets within this network and identify key players in the transition to fetal-like HSPCs, the authors considered the following criteria: (1) regulatory regions bound by smarca5; (2) reduced chromatin accessibility in the smarca5 mutant; (3) lower expression levels in the smarca5 mutant; (4) presence of hematopoietic transcription factor binding motifs. One of the most compelling targets that came out of this analysis was bcl11ab, an important regulator of hemoglobin switching.⁹  Even more striking was that overexpression of bcl11ab could partially rescue the smarca5 mutant phenotype, suggesting it is a critical downstream target of smarca5 in regulating the transition to fetal-like hematopoiesis.

This study demonstrates the persistent strength of the zebrafish model to provide powerful genetic tools to address fundamental in vivo biological questions. However, to have broad implications for the in vitro differentiation of human long-term repopulating HSCs from embryonic or induced pluripotent stem cells, the authors wanted to show that smarca5 has a conserved role in development of mammalian fetal-like HSPCs. They found that SMARCA5 is enriched in human fetal liver hematopoietic cells based on published single-cell RNA-seq data.¹⁰  They went on to validate Smarca5 function using colony forming unit assays and small interfering RNA knockdown in mouse fetal liver HSPCs. The Smarca5 knockdown HSPCs produced fewer granulocyte-erythrocyte-monocyte-megakaryocyte (GEMM), and granulocyte-macrophage (GM) colonies.

Although smarca5, together with nucleolin, clearly has a role in increasing the chromatin accessibility of key hematopoietic genes during the transition to fetal-like HSPCs, it is not the only factor. The authors suggest in experiments not shown that smarca5 is required but not sufficient to complete the transition. This leaves open the question of what other factors would enable exponential expansion of fetal-like HSPCs in vitro for clinical applications.