Journal: Communications Biology

Article Title: SATB1 prevents immune cell infiltration by regulating chromatin organization and gene expression of a chemokine gene cluster in T cells

doi: 10.1038/s42003-024-07021-8

Figure Lengend Snippet: a Western blot shows the efficient depletion of SATB1 in CD4 + T cells. b A representative mouse from each group is photographed (6–16 weeks male mice). c Statistical comparison of quantified body weight among Ctrl and Satb1 cKO mice (6–16 weeks male mice, n = 10 for each group). Error bars indicate Mean ± SD and each dot represents a mouse. Statistical significance is measured via paired Student’s t-tests and is presented as follows: *** p < 0.005. d Volcano plot shows the 365 significantly down-regulated (log2FC < −1 and padj < 0.05) and 794 significantly up-regulated (log2FC > 1 and padj < 0.05) genes in activated naive CD4 + T cells of Ctrl and Satb1 cKO. About 6–16 weeks male mice were used in this experiment and each group had 3 independent biological replicates. e Dot plots indicate the top 5 most enriched BP (biological process) GO terms in up-regulated genes identified in ( a ). f Heatmaps of Ccl3 , Ccl4, Ccl5, Ccl6 and Ccl9 genes. Each column shows the scaled gene expression in an RNA-Seq replicate, each row represents a gene. g Violin plots of differentially expressed genes in each chemokine family. Statistical significance is measured via unpaired Student’s t-tests and is presented as follows: * p < 0.05, ** p < 0.01, *** p < 0.005. h , i Immunohistochemical stainings for CD4 and CCL3 in lung tissues for each group. The representative image shows the lung infiltrating immune cells expressing CD4 and CCL3. j Flow cytometric sorting strategy for CD4 + T cells in lung tissues. The lung tissues were digested into single-cell suspension and stained CD4 antibodies for flow cytometric sorting. k Protein levels of chemokine CCL3 in lung-infiltrating T cells (sorted from ( g )) were measured using ELISA ( n = 3). Statistical significance is measured via unpaired Student’s t-tests and is presented as *** p < 0.005.

Article Snippet: CCL3 protein level was determined using Mouse CCL3/MIP-1α ELISA Kit (Liankebio, EK261) according to the manufacturer’s instructions.

Techniques: Western Blot, Comparison, Gene Expression, RNA Sequencing, Immunohistochemical staining, Expressing, Suspension, Staining, Enzyme-linked Immunosorbent Assay

Journal: Communications Biology

Article Title: SATB1 prevents immune cell infiltration by regulating chromatin organization and gene expression of a chemokine gene cluster in T cells

doi: 10.1038/s42003-024-07021-8

Figure Lengend Snippet: a Principle component analysis (PCA) of the samples used for H3K27ac CUT&Tag analysis. b Heatmap displaying the correlation of H3K27ac CUT&Tag experiments replicates. c Volcano plot showing 8561 differential H3K27ac peaks (4694 up-regulated in red and 3861 down-regulated in blue) between Satb1 cKO and control mice. d Boxplot compares expression changes for genes adjacent to H3K27ac peaks. Genes with peaks of H3K27ac in their promoter regions are classified as genes adjacent to H3K27ac peaks. The median values (-0.195, 0.109, 0.016) are displayed on top of the graphs. e H3K27ac signals on the gene cluster sites of Ccl3, Ccl4, Ccl5, Ccl6 , and Ccl9 are visualized by IGV software. No significant change was observed in the H3K27ac levels at these sites, while a noticeable enhancer site was identified in the middle of the gene loci of Ccl3 and Ccl6 , named as Ccl -Enhancer, which was labeled in the gray box.

Article Snippet: CCL3 protein level was determined using Mouse CCL3/MIP-1α ELISA Kit (Liankebio, EK261) according to the manufacturer’s instructions.

Techniques: Control, Expressing, Software, Labeling

Journal: Communications Biology

Article Title: SATB1 prevents immune cell infiltration by regulating chromatin organization and gene expression of a chemokine gene cluster in T cells

doi: 10.1038/s42003-024-07021-8

Figure Lengend Snippet: a Hi-C heatmaps at 10 kb resolution show changed chromatin interactions at Ccl3, Ccl4, Ccl5, Ccl6 , and Ccl9 gene loci in thymic T cells. CUT&Tag profiles show the SATB1 and CTCF signals in control and Satb1 cKO thymocyte cells. The Ccl -Enhancer was labeled in the gray box. b Diagram depicting the regions of subTAD and adjacent and boxplot showing the interaction frequencies of different regions indicated in ( a ). The adjacent region was set as an internal reference. Error bars indicate Mean ± SD. Statistical significance is measured via unpaired Student’s t-tests and is presented as follows: * p < 0.05, ** p < 0.01, *** p < 0.005. c The diagram depicted the 3C-qPCR experiment design and the 3C-qPCR experiment was used to validate the enhancer-promoter interaction of Ccl3, Ccl4, Ccl5, Ccl6 , and Ccl9 genes in both control and Satb1 cKO naive CD4 + T cells. A negative control was also detected. Error bars indicate Mean ± SD. Statistical significance is measured via unpaired Student’s t-tests and is presented as follows: * p < 0.05, ** p < 0.01, *** p < 0.005. d dCas9 experiment was performed to validate the function of enhancer-promoter interaction in the regulation of gene expression for Ccl3, Ccl4, Ccl5, Ccl6 , and Ccl9 . Error bars indicate Mean ± SD. Statistical significance is measured via unpaired Student’s t-tests and is presented as follows: * p < 0.05, ** p < 0.01, *** p < 0.005.

Article Snippet: CCL3 protein level was determined using Mouse CCL3/MIP-1α ELISA Kit (Liankebio, EK261) according to the manufacturer’s instructions.

Techniques: Hi-C, Control, Labeling, Negative Control, Gene Expression

Journal: Communications Biology

Article Title: SATB1 prevents immune cell infiltration by regulating chromatin organization and gene expression of a chemokine gene cluster in T cells

doi: 10.1038/s42003-024-07021-8

Figure Lengend Snippet: SATB1 inhibited Ccl gene expression by restraining enhancer-promoter interaction. The loss of SATB1 led to the emergence of a new chromatin domain encompassing the Ccl3, Ccl4, Ccl5, Ccl6 , and Ccl9 genes and a distal enhancer, which possibly mediated by CTCF, resulting in increased contacts between the enhancer and all five chemokine genes, thus inducing autoimmunity.

Article Snippet: CCL3 protein level was determined using Mouse CCL3/MIP-1α ELISA Kit (Liankebio, EK261) according to the manufacturer’s instructions.

Techniques: Gene Expression

Journal: Communications Biology

Article Title: SATB1 prevents immune cell infiltration by regulating chromatin organization and gene expression of a chemokine gene cluster in T cells

doi: 10.1038/s42003-024-07021-8

Figure Lengend Snippet: Primers for RT-qPCR

Article Snippet: CCL3 protein level was determined using Mouse CCL3/MIP-1α ELISA Kit (Liankebio, EK261) according to the manufacturer’s instructions.

Techniques:

Journal: Communications Biology

Article Title: SATB1 prevents immune cell infiltration by regulating chromatin organization and gene expression of a chemokine gene cluster in T cells

doi: 10.1038/s42003-024-07021-8

Figure Lengend Snippet: Primers for 3C-qPCR

Article Snippet: CCL3 protein level was determined using Mouse CCL3/MIP-1α ELISA Kit (Liankebio, EK261) according to the manufacturer’s instructions.

Techniques: Control, Negative Control