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STAR Protocols · 2022 · IF 0

Protocol for isolation and ATAC-seq library construction of zebrafish red blood cells

Yanyan Ding,Feng Liu

Summary Understanding chromatin dynamics in red blood cells (RBCs) is critical for exploring the differentiation process and homeostasis maintenance during erythropoiesis. Here, we describe a protocol for isolation of zebrafish erythrocytes labelled with gata1 :dsRed by fluorescence-activated cell sorting. We detail steps for ATAC-seq library construction from the isolated RBCs and describe how to analyze the quality of the library. The library can then be used to assay genome-wide chromatin accessibility in these RBCs. For complete details on the use and execution of this protocol, please refer to Ding et al. (2021). 1

研究领域: 基因组学 · 分子生物学 · 血液学

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DEVELOPMENT · 2024 · IF 3.7

The unique functions of Runx1 in skeletal muscle maintenance and regeneration are facilitated by an ETS interaction domain

Yu Meng,Thorner Konrad,Parameswaran Sreeja,Wei Wei,Yu Chuyue,Lin Xinhua,Kopan Raphael,Hass Matthew R.

The conserved Runt-related (RUNX) transcription factor family are master regulators of developmental and regenerative processes. Runx1 and Runx2 are expressed in satellite cells (SC) and in skeletal myotubes. Conditional deletion of Runx1 in adult SC negatively impacted self-renewal and impaired skeletal muscle maintenance even though Runx2 expression persisted. Runx1 deletion in C2C12 cells that retain Runx2 expression identified unique molecular functions of Runx1 that cannot be compensated by Runx2. The reduced myoblast fusion in vitro caused by Runx1 loss was due in part to ectopic expression of Mef2c, a target repressed by Runx1 . Structure-function analysis demonstrated that the Ets-interacting MID/EID region of Runx1, absent from Runx2, is critical to Runx1 myoblast function and for Etv4 binding. Analysis of ChIP-seq datasets from Runx1 (T-cells, muscle) versus Runx2 (preosteoblasts) dependent tissues identified a composite Ets:Runx motif enriched in Runx1 -dependent tissues. The Ets:Runx composite motif was enriched in peaks open exclusively in ATAC-seq datasets from WT cells compared to ATAC peaks unique to Runx1KO cells. Thus, engagement of a set of targets by the RUNX1/ETS complex define the non-redundant functions of Runx1 .

研究领域: 分子生物学 · 细胞生物学 · 转录因子 · 肌肉再生

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Protocol for isolation and ATAC-seq library construction of zebrafish red blood cells

The unique functions of Runx1 in skeletal muscle maintenance and regeneration are facilitated by an ETS interaction domain