Terzi Çizmecioğlu Nihal
Department of Biological Sciences, Middle East Technical University, Faculty of Arts and Sciences, Ankara, Turkey.
J Turk Ger Gynecol Assoc. 2023 Sep 7;24(3):187-196. doi: 10.4274/jtgga.galenos.2023.2023-7-5.
Proper deactivation of the pluripotency network and activation of a lineage-specific gene expression program are critical for mouse embryonic stem cell (mESC) differentiation. This is achieved by the coordinated action of transcription and chromatin factors. Our previous work identified ARID4B as a critical chromatin factor for mesoderm and endoderm differentiation. As part of a histone deacetylase complex, ARID4B plays a role in transcriptional suppression of its direct targets. Here, we investigated the mechanism of ARID4B function in mESC differentiation by focusing on genes and pathways that are upregulated in its absence.
We analyzed transcriptomic results of wild-type and arid4bΔ endoderm or mesoderm differentiated cells through integrative genomics viewer and ingenuity pathway analysis. We performed real-time quantitative polymerase chain reaction for selected genes. To understand pathway activation, we performed Western blot for candidate proteins during the time-course of differentiation. We also analyzed H3K4me3, H3K27me3 and H3K27Ac ChIP-seq results to understand changes in the chromatin environment.
Interferon-related genes were activated in arid4bΔ mESCs and endoderm or mesoderm directed cells. Consistent with this, higher phosphorylated STAT1 levels were found in arid4bΔ mESCs while a related phosphorylated STAT3 was unchanged. Finally, we observed a significant increase in H3K4me3 around interferon-related distal gene regulatory regions with a combination of either upregulation of H3K27Ac level or downregulation of H3K27me3 level.
These results provide evidence that ARID4B is involved in the suppression of interferon-related genes in mESCs and during meso/endoderm differentiation through modulation, mainly of H3K4me3. This regulation might be important for successful mESC differentiation.
多能性网络的适当失活和谱系特异性基因表达程序的激活对于小鼠胚胎干细胞（mESC）分化至关重要。这是通过转录因子和染色质因子的协同作用实现的。我们之前的工作确定ARID4B是中胚层和内胚层分化的关键染色质因子。作为组蛋白去乙酰化酶复合物的一部分，ARID4B在其直接靶标的转录抑制中发挥作用。在这里，我们通过关注在其缺失时上调的基因和途径，研究了ARID4B在mESC分化中的功能机制。
我们通过综合基因组浏览器和 Ingenuity 通路分析，分析了野生型和 arid4bΔ内胚层或中胚层分化细胞的转录组结果。我们对选定的基因进行了实时定量聚合酶链反应。为了了解通路激活情况，我们在分化的时间进程中对候选蛋白进行了蛋白质印迹分析。我们还分析了H3K4me3、H3K27me3和H3K27Ac ChIP-seq结果，以了解染色质环境的变化。
干扰素相关基因在arid4bΔ mESC以及内胚层或中胚层定向细胞中被激活。与此一致的是，在arid4bΔ mESC中发现较高的磷酸化STAT1水平，而相关的磷酸化STAT3没有变化。最后，我们观察到在干扰素相关的远端基因调控区域周围，H3K4me3显著增加，同时伴有H3K27Ac水平上调或H3K27me3水平下调。
这些结果提供了证据，表明ARID4B通过主要调节H3K4me3，参与了mESC以及中胚层/内胚层分化过程中干扰素相关基因的抑制。这种调节可能对mESC的成功分化很重要。
