Journal: Nucleic Acids Research

Article Title: Dynamic regulation of chromatin organizer SATB1 via TCR-induced alternative promoter switch during T-cell development

doi: 10.1093/nar/gkaa321

Figure Lengend Snippet: Cell type-specific expression of Satb 1 transcript variants during thymocyte development. ( A ) Schematic diagram depicting the various developmental stages of thymocytes. The relevant cell surface markers are indicated. The color code of various subpopulations of the cells is the same as the bars of relative expression values in ( B ). ( B and C ) Quantitative RT-qPCR analyses were performed to measure the expression levels of total Satb1 mRNA and Satb 1 transcript variants in various developmental stages of thymocytes such as DN, DP, immature CD4SP and mature CD4SP, and DP, total CD4SP and total CD8SP. 18S rRNA expression was used as endogenous control for normalization. The presented data are from three independent experiments and is shown as means of ± SEM. P -values were calculated using ANOVA or unpaired two-tailed Student's t -test. * P < 0.05; ** P < 0.01; *** P < 0.001. ( D ) RNA-seq analysis of CD3 low DP and CD4 SP thymocytes was performed using publicly available datasets. Sashimi plot analysis represents the alternative splicing events of Satb1 alternative first exons between DP and CD4SP thymocytes. ( E ) RNA-seq based gene expression values (FPKM) corresponding to expression of the alternatively spliced variants of Satb1 and total SATB1 in DP and CD4 SP thymocytes are presented in the two histograms ( F ) Expression of SATB1 protein in CD4 + CD8 + DP, immature CD4 + CD24 + SP and mature CD4 + CD24 − SP thymocytes was analyzed using flow cytometry and the mean florescence intensities (MFI) of SATB1 in these three populations are shown; n = 3, P < 0.01. The percentage change from DP to CD4 immature is calculated using the formula: 100*[MFI(CD4imm)-MFI(DP)]/MFI(DP). Similarly, it was calculated for the other populations. ( G ) Western blotting to compare SATB1 protein levels in isolated populations of DN, DP, CD4 and CD8 thymocytes (panel on the left). β-ACTIN was used as an endogenous control. Densitrometric analysis was performed using Fiji (ImageJ v.1.52a), and normalization was achieved using respective ACTIN signals (histogram on the right); Statistical analysis was performed using ANOVA (Graphpad v8) n = 3, * P < 0.05, ** P < 0.01.

Article Snippet: Spleen from six-week-old mice were used for enrichment of naïve CD4 + T cells by magnetic sorting using the mouse CD4 + T-cell enrichment kit (BD Biosciences) followed by FACS sorting.

Techniques: Expressing, Quantitative RT-PCR, Two Tailed Test, RNA Sequencing Assay, Flow Cytometry, Western Blot, Isolation

Journal: Nucleic Acids Research

Article Title: Dynamic regulation of chromatin organizer SATB1 via TCR-induced alternative promoter switch during T-cell development

doi: 10.1093/nar/gkaa321

Figure Lengend Snippet: TCR signal induces Satb1 promoter switch in CD4SP thymocytes. ( A ) Flow cytometry analysis of SATB1 expression in TCR signaling active DP and CD4SP thymocytes. Thymocytes were stained with anti-CD4, anti-CD8, anti-CD69 and anti-SATB1 antibodies and FACS analyses were performed using BD FACS canto II. ( B ) SATB1 expression in CD69 + DP, CD69 + CD4SP and CD69 − CD4SP thymocytes is shown by histogram analysis using FlowJo V10.5.2. ( C ) CD69 + DP, CD69 + CD4SP, CD69 − CD4SP thymocyte populations were FACS sorted and cells were used for RNA extraction and cDNA synthesis. qRT-PCR was performed to estimate the levels of total Satb 1 transcripts as well as its transcript variants. The color key is same as used in (B). The represented data are from three independent experiments and is shown as means of ± SEM. P -values were calculated using one-way ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Spleen from six-week-old mice were used for enrichment of naïve CD4 + T cells by magnetic sorting using the mouse CD4 + T-cell enrichment kit (BD Biosciences) followed by FACS sorting.

Techniques: Flow Cytometry, Expressing, Staining, RNA Extraction, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Dynamic regulation of chromatin organizer SATB1 via TCR-induced alternative promoter switch during T-cell development

doi: 10.1093/nar/gkaa321

Figure Lengend Snippet: Lineage-specific chromatin dynamics of Satb1 alternative promoter regions during thymic T-cell development from precursor cells. ( A ) We used publicly available ATAC-seq data from HSCs, MPPs, CLPs, B cells and T cells to analyze the state of chromatin accessibility at the alternative promoters of the Satb1 gene locus in these cell types. Satb 1 alternative promoter regions (P1, P2, P3 and P4) are marked by rectangular boxes at the Satb1 gene locus. ( B ) We analyzed the publicly available ATAC-seq datasets performed in various T-cell developmental stages; DN1, DN2a, DN2b, DN3, DN4, DP, CD4SP and CD8SP to assess the chromatin dynamics at the alternative promoters of the Satb1 gene locus. We also used ATAC-seq data from naïve CD4 and CD8 populations alongside the developmental stages. The distinct Satb 1 alternative promoter regions (P1, P2, P3 and P4) are marked by rectangular boxes at the Satb 1 gene locus.

Article Snippet: Spleen from six-week-old mice were used for enrichment of naïve CD4 + T cells by magnetic sorting using the mouse CD4 + T-cell enrichment kit (BD Biosciences) followed by FACS sorting.

Techniques:

Journal: Nucleic Acids Research

Article Title: Dynamic regulation of chromatin organizer SATB1 via TCR-induced alternative promoter switch during T-cell development

doi: 10.1093/nar/gkaa321

Figure Lengend Snippet: TCF1 directly regulates the SATB1 expression in CD4SP thymocytes during T-cell development. ( A ) A TCF1/LEF1 canonical DNA-binding motif was identified in the P2 promoter region of Satb1 using MEME-Suite (Version 5.1.1, http://meme-suite.org/tools/meme ). ( B ) TCF1 ChIP was performed in sorted DP and CD4SP thymocytes, followed by qRT-PCR analysis using primers spanning the TCF1/LEF1 binding site on P2 promoter. Two different antibody clones were used (depicted as #1 and #2 in the figure axis) to assess the antibody-factor specificity. Different control regions were used to ascertain whether TCF1 specifically binds to the Satb 1 promoter regions, labeled C1 and C2, at 1000 and 1500 bp upstream of P2, respectively. ( C ) qRT-PCR analysis of expression profile of total Satb 1, P1, P2, P3 and P4 variants in CD4SP thymocytes along with other sorted T-cell developmental stages from wild-type and TCF1 KO mice. Hprt gene expression was used as endogenous control. The represented data are from three independent experiments and is shown as means of ± SEM. P -values were calculated using one-way ANOVA or. * P < 0.05; ** P < 0.01; *** P < 0.001. ( D ) Total thymocytes were isolated from either WT or TCF1 KO mice and stained with anti-CD4, anti-CD8 and anti-SATB1 antibodies. FACS analyses were performed using BD FACS canto II with the indicated gating. ( E ) Flow cytometry analysis shows the expression of SATB1 in thymocyte developmental stages; DN, DP, CDSP and CD8SP from wild-type and TCF1 KO mice as indicated by black and red color curved lines respectively. Solid gray peaks indicate the staining controls. ( F ) Satb 1 P2 promoter was cloned in pGL3 basic vector and subjected to dual luciferase activity assay in the background of TCF1 overexpression in HEK293 cells. Renilla was used as an endogenous control. The represented data are from three independent experiments and is shown as means of ± SEM. P -values were calculated using the unpaired two-tailed Student's t -test. * P < 0.05. ( G ) A schematic representation depicting how TCF1 acts upstream of SATB1 during differentiation of CD4SP from DP in thymus.

Article Snippet: Spleen from six-week-old mice were used for enrichment of naïve CD4 + T cells by magnetic sorting using the mouse CD4 + T-cell enrichment kit (BD Biosciences) followed by FACS sorting.

Techniques: Expressing, Binding Assay, Quantitative RT-PCR, Clone Assay, Labeling, Isolation, Staining, Flow Cytometry, Plasmid Preparation, Luciferase, Activity Assay, Over Expression, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Dynamic regulation of chromatin organizer SATB1 via TCR-induced alternative promoter switch during T-cell development

doi: 10.1093/nar/gkaa321

Figure Lengend Snippet: TCR signal mediates Satb1 promoter switch in the peripheral CD4 + T cells. ( A ) Naïve CD4 + T cells were isolated from spleen of six-week-old C57BL/6 mice and were subjected to TCR activation by culturing in the presence of plate bound anti-CD3 and soluble anti-CD28 for 48 h. Cells were harvested and used for qRT-PCR, western blotting and FACS analysis. Naïve and TCR-activated cells were processed for western blotting which was performed using anti-SATB1 and anti-tubulin. Two replicates are shown (Lanes 1–2 and 3–4) to demonstrate the robustness of TCR signaling mediated upregulation of SATB1 in CD4 T cells. ( B ) FACS analysis were performed to generate the histograms of SATB1 and Nur77 by staining cells with anti-CD4, anti-CD8, anti-CD25, anti-SATB1 and anti-Nur77. ( C ) Naïve and TCR-activated cells were used for RNA extraction and cDNA synthesis. Next, quantitative RT-PCR (qRT-PCR) analyses were performed to measure the expression of total Satb1 and its transcript variants. Expression of 18S rRNA was used as an endogenous control. The represented data are from three independent experiments and is shown as means of ± SEM. P -values were calculated using unpaired two-tailed Student's t -test. * P < 0.05; ** P < 0.01.

Article Snippet: Spleen from six-week-old mice were used for enrichment of naïve CD4 + T cells by magnetic sorting using the mouse CD4 + T-cell enrichment kit (BD Biosciences) followed by FACS sorting.

Techniques: Isolation, Activation Assay, Quantitative RT-PCR, Western Blot, Staining, RNA Extraction, Expressing, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Dynamic regulation of chromatin organizer SATB1 via TCR-induced alternative promoter switch during T-cell development

doi: 10.1093/nar/gkaa321

Figure Lengend Snippet: A schematic model depicting TCR signal-mediated Satb1 alternative promoter switch in developing thymocytes. During thymic T-cell development, DP thymocytes are generated as a result of pre-TCR signaling at DN4 stage. At the DP stage of development, TCRα gene rearrangement takes place and DP thymocytes begin to receive TCR signaling. Persistent or stronger TCR signaling at the DP stage leads to their differentiation toward CD4 + T cells, whereas cessation or weaker TCR signaling leads to CD8 + T-cell differentiation. During T-cell development, DP thymocytes express P1 (P1S and P1L) and P4 transcript variants along with the constitutively active P3 variant. Upon TCR signal mediated differentiation of DP into CD4SP, CD4 + T cells switch to increased expression of P2 along with P3 variant, presumably due to direct binding by the TF TCF1. Since Satb 1 transcript variants exhibit differential translatability, the combinatorial expression of Satb1 transcript variants plays a key role in maintaining SATB1 protein levels.

Article Snippet: Spleen from six-week-old mice were used for enrichment of naïve CD4 + T cells by magnetic sorting using the mouse CD4 + T-cell enrichment kit (BD Biosciences) followed by FACS sorting.

Techniques: Generated, Cell Differentiation, Variant Assay, Expressing, Binding Assay