1. Volume 20, Issue 6, Pages 719-733 (June 2004)
The Chromatin Remodeler Mi-2β Is Required for CD4 Expression and T Cell Development 
Christine J Williams, Taku Naito, Pablo Gómez-del Arco, John R Seavitt, Susan M Cashman, Beverly De Souza, Xiaoqing Qi, Piper Keables, Ulrich H Von Andrian, Katia Georgopoulos 
Immunity 
Volume 20, Issue 6, Pages (June 2004)
DOI: /j.immuni

2. Figure 1 Lckpr-Cre-Induced Deletion of Mi-2β in Developing T Cells
(A) For conditional inactivation strategy, exons 12–21 of Mi-2β were flanked by LoxP sites (black triangles); the Neo marker was flanked by Frt sites (white triangles). Flp recombinase excises the Neo gene from the targeting construct and generates the Mi-2βLoxPF allele. Lckpr-Cre recombinase induced deletion of the Mi-2β ATPase domain in thymocytes, generating the Mi-2βΔF allele, or if introduced before Flp, the Mi-2βΔ (Neo+) allele.
(B) Southern blot analysis of Mi-2β deletion efficiency. Genomic DNA from tail, thymus, lymph nodes (LN), and LN T cells (LN T) was extracted from wt (Mi-2β+/+), heterozygous (Lckpr-Cre/Mi-2β+/LoxPF), or Mi-2β-deficient (Lckpr-Cre/Mi-2βΔ/LoxPF) mice. EcoRI-digested DNA was probed as indicated in (A). Digestion fragments from both the + and LoxPF alleles migrated at 8.5 kb; the Δ allele (Neo+) migrated at 5 kb. The 8.5 kb LoxP fragment was reduced to 3.5 kb after Cre-mediated excision of exons 12–21 of Mi-2β (ΔF). Deletion efficiency was >95% in thymus and varied from 13%–45% in total LN and 15%–65% in LN T cells.
(C) Reduction of full-length Mi-2β mRNA in thymic subpopulations. RNA was extracted from KO and Mi-2β+/+ thymic subpopulations based on CD4 and CD8 expression (Figure 2B). RT-PCR was performed with primers spanning exons 11–13 (within the deleted region) or exons 30–34 (downstream of the deletion region) of the Mi-2β gene, exons 29–32 of the Mi-2α gene, or β-actin gene as a control. Two dilutions of cDNA (neat, 1:5) were used for each sample.
(D) Western blot analysis of thymus nuclear extracts from wt (Mi-2β+/+), heterozygote (Mi-2β+/ΔF), and Mi-2β-deficient (Mi-2βΔF/ΔF) littermates. The antibodies used are indicated. YY1 protein levels are shown as a loading control.
(E) Western blot analysis of Mi-2β expression in whole-cell extracts from Mi-2β+/+ (wt) or Mi-2βΔF/ΔF (KO) thymus subpopulations. Thymocytes were sorted into the indicated populations as described in (C). Ikaros and YY1 protein levels are also displayed.
Immunity  , DOI: ( /j.immuni )

3. Figure 2 Mi-2β Inactivation Alters Thymocyte Differentiation
(A) Schematic representation of developmental transitions and gene expression during T cell development in the thymus. Expression of CD4 and CD8 coreceptors; relative levels of cell surface markers TCR, CD5, and HSA; and pro- and antiapoptotic genes Fas and Bcl-2 are indicated.
(B) Mi-2β deletion leads to dysregulation of CD4 expression in DP thymocytes. Three color FACS analysis of thymus profiles from 5 weeks wt and Mi-2βΔF/ΔF littermates are shown. Dot plots display CD4 versus CD8 expression and numbers beside each box indicate the percentage of cells in each subpopulation. Thymus cellularity is indicated. Thymocytes from wt (green) or KO (red) littermates were sorted into five populations (DN, DP, CD8+CD4lo, CD8+CD4−, CD4+). Expression of TCR and HSA in the five populations is displayed as single color histograms. Dotted lines representing mean fluorescence intensity (MFI) in each sample are included for comparison. Two dotted lines appear overlaying the wt CD8+CD4− histogram. These represent the CD8+ SP (HSAlo, TCRhi) and ISP (HSAhi, TCRlo) populations that are normally found within this subset. Note that the MFI for HSA expression in the KO CD8+CD4− population falls between the wt ISP and wt CD8+ SP peaks and is similar to that of DP.
(C) Expression of CD4 protein and mRNA is reduced in Mi-2βΔF/ΔF thymocytes. CD4 surface expression in three sorted populations of thymocytes wt or KO littermates is displayed as single color histogram overlays (wt/green; KO/red). Dotted lines represent MFI. RNA was prepared from the same sorted thymocyte populations, then RT-PCR was performed with primers specific for the CD4 gene. β-actin level is shown as a control.
(D) Mi-2β-deficient CD8+ thymocytes resemble DP in their expression of Fas and Bcl-2. RT-PCR was performed with primers specific for Fas and Bcl-2 using RNA prepared from four sorted subpopulations (DN, DP, CD8+CD4−, CD4+) and thymus from wt and KO littermates.
Immunity  , DOI: ( /j.immuni )

4. Figure 3 Effects of Mi-2β Deficiency on the DN to DP Transition
(A) Bar graph summary of cell numbers and percentages of total thymocytes and DN populations in KO (n = 24) versus wt (n = 26).
(B) DN1-DN4 profiles in the adult wt and KO thymus. Lin−/Thy1+ thymocytes were examined for expression of CD44 and CD25, revealing an increase in the mutant DN4 population.
(C) DN3 (CD44−/CD25+) and DN4 (CD44−/CD25−) populations were sorted and analyzed for expression of IC-TCRβ, cell cycle profile (propidium iodide), and apoptosis (AnnexinV/propidium iodide). mRNA was prepared from the same sorted subsets, and expression of Mi-2β (exons 11–13), Mi-2α, and β-actin was determined.
(D) Temporal block at the DN stage of differentiation in neonatal thymus of Mi-2β KO. In contrast to wt, the majority of mutant thymocytes show a high FSC reflecting an immature blasting state. Further analysis of DN thymocytes (as in [B]) revealed an accumulation of DN4 (CD44−/CD25−) cycling cells in the KO.
Immunity  , DOI: ( /j.immuni )

5. Figure 4 T Cell Maturation in the Absence of Mi-2β
(A) Mi-2β-deficient mice exhibit a reduction in peripheral T cells. CD4 and CD8 staining on peripheral LN from 6-week-old wt and KO littermates is shown. The total LN cell count for each animal and the percentage of cells in each quadrant is shown. An arrowhead points to the MFI of CD4 expression in each animal. Arrowheads indicating that CD8 fluorescence is unchanged are included as a control.
(B) Mi-2β-deficient T cells are hypoproliferative. Since Mi-2β deletion was <100% in peripheral T cells, Lckpr-Cre/Mi2βLoxPF/LoxPF mice were crossed to the LacZ/EG indicator strain in which cells actively expressing Cre recombinase are marked by GFP expression. GFP+ LN cells were then sorted that were either Lckpr-Cre/Mi2β+/+ (wt) or Lckpr-Cre/Mi2βLoxPF/LoxPF (KO). Mi-2β deletion efficiency in Lckpr-Cre/Mi2βLoxPF/LoxPF GFP+ T cells (>95%) is displayed in the PCR panel on the right. The GFP− T cell pool also contains a fraction of Mi-2βΔF/ΔF T cells, suggesting that GFP expression in the indicator line lags behind Cre activity. LN or GFP+ T cells were activated in vitro with either α-CD3 or PMA and ionomycin. Bar graphs of the scintillation counts represent the relative proliferation index for the wt or KO cells.
(C) Inability of activated Mi-2βΔF/ΔF T cells to go through normal cell divisions is revealed by FACS analysis of a combination of CFSE labeling and cell size (FSC). The overlay of CFSE staining of wt versus KO T cells at 48 hr after activation shows a difference in the fraction of cycling cells.
(D) Induction (0 to 48 hr) of activation markers (CD25 and CD69) in Mi-2βΔF/ΔF T cell activation cultures.
Immunity  , DOI: ( /j.immuni )

6. Figure 5
Mi-2β Is Required for Developmentally Regulated Expression of a CD4 Transgenic Reporter
Decreased expression of an exogenous GFP-expressing CD4 transgene in Mi-2β-deficient thymocytes. Five thymocyte subpopulations were identified based on CD4 and CD8 expression in the wt and KO thymus (see gates, top). Expression levels of the T-GFP-CD4 transgene (Manjunath et al., 1999) are shown as overlaid histograms within these subsets (wt, green; KO, red). Expression of a Rosa26-YFP transgene in KO thymocyte subpopulations is provided as a control for specificity of the Mi-2β deficiency effect on expression of the CD4-transgene. Dotted lines indicate the MFI in each of the subpopulations in the T-GFP-CD4 and Rosa26-YFP transgenes.
Immunity  , DOI: ( /j.immuni )

7. Figure 6
Mi-2β Association and Histone H3 Acetylation of CD4 Regulatory Elements
(A) Schematic representation of the CD4 locus. Exons around the CD4 locus are shown in the upper half, and the known CD4 regulatory elements are indicated in the bottom half of the gene.
(B) Results of α-Mi-2β and α-AcH3 ChIP. Chromatin samples from wt (upper panels) or Mi-2β KO (lower panels) thymocytes were immunoprecipitated using control mouse IgG, α-Mi-2β, or α-AcH3. Coprecipitated DNA was analyzed by multiplex PCR. In each panel, the upper bands are from the region of interest, whereas the bottom bands (asterisks) are internal controls from an irrelevant genomic region. Fold enrichment relative to IgG ChIP was calculated as described (Harker et al., 2002). Relatively strong enrichment of the proximal enhancer and to a lesser extent, the promoter, was obtained with α-Mi-2β and α-AcH3. Enrichment was diminished in the KO sample, indicating dependence on the presence of Mi-2β.
(C) Graphical presentation of the calculated fold enrichment for α-Mi-2β ChIP from (B). The results for the wt and KO are plotted with black and gray boxes, respectively.
(D) Fold enrichment for α-AcH3 ChIP from (B) is plotted as (C).
Immunity  , DOI: ( /j.immuni )

8. Figure 7
Mi-2β Promotes Recruitment of HEB and p300 to the CD4 Enhancer
(A) p300 HAT association with the CD4 regulatory regions. ChIP was performed using α-p300. The results of the multiplex-PCR analyses for the proximal enhancer, promoter, exon 10, LCR, and 3′ enhancer are shown. Asterisks indicate the internal control. Calculated fold enrichment of p300 association with the CD4 regulatory region is shown on the right as a bar graph. α-p300 gave strong enrichment at the first half of the proximal enhancer, which was abolished in the KO.
(B) HEB association with the CD4 enhancer is Mi-2β dependent. ChIP analysis with α-HEB was performed as described in (A). Given the very strong association of HEB with the enhancer, PCR reactions for the target and internal control were performed separately and a 1:9 dilution of the template was used for target amplification.
(C) Immunoprecipitations of thymocyte nuclear extracts with α-p300 (+ and − ethidium bromide), α-HEB, or IgG control and of thymocyte nuclear extracts expressing flag-tagged Mi-2β with α-flag followed by elution and Western blotting with α-p300, α-Mi-2β, α-HEB, and α-HDAC2 reveal a physical association between HEB, p300, and Mi-2β but not HDAC2.
Immunity  , DOI: ( /j.immuni )

9. Figure 8
The Role of Mi-2β in T Cell Development and a Model on CD4 Gene Regulation
(A) A summary of the effects of Mi-2β depletion on T cell development. X indicates a block in DN differentiation, defect in CD4 gene expression, and a defect in antigen receptor-mediated proliferative expansion of SP T cells. “+/−” shows severe reduction in CD4 expression on DP and a downward arrow lower levels of CD4 expression on CD4 SP T cells.
(B) Mi-2β and p300 interacting with HEB are recruited to HEB cognate sites (E box sites) on the CD4 enhancer. Mi-2β promotes chromatin fluidity in an ATP-dependent fashion and stabilizes HEB binding. The presence of p300 in complex with either HEB or Mi-2β or both induces local histone hyperacetylation (flags) that locks chromatin in an open configuration. Interactions between the activated enhancer and the CD4 promoter (broad arrow) stabilize recruitment of basal transcription factors resulting in CD4 gene expression.
Immunity  , DOI: ( /j.immuni )