Supplemental Data for:

Chi et al., Immunity 19, pp. 169–182

 

Supplemental Figure S1. Thy1.2 Negative Cells, Marking Non-T Cells, Did Not Significantly Contaminate DN4 Population

 

Cells were stained with antibodies as indicated. Debris and potential red blood cells were gated out prior to analysis based on their small forward scatter. The results indicate that Thy1.2 negative cells constitute about 10% of total DN cells (column 1), but less than 50% of these cells were CD25^-CD44^- (column 4). As only the CD25^-CD44^- cells were scored as the DN4 cells, the Thy1.2 negative cells therefore constituted only 5% and 14% of total DN4 population in WT and Brg^F/-;Cre mice, respectively. This estimate is consistent with the fact that there is little difference in the DN4 abundance in the Thy1.2 positive versus negative subsets of DN cells (compare columns 1 and 2). That a significant percentage of Thy1.2 negative cells were CD25^-CD44⁺ characteristic of DN1 cells suggests that some DN1 cells has not yet expressed Thy1.2 at such an early stage of T cell development.

 

Supplemental Figure S2. A Hydraulic Model for the Accumulation of Bcl-xL Rescued Brg-Deficient Cells

 

A subset of DN2, DN3, and all of the DN4 cells delete Brg during thymic development in Brg^F/-;Cre mice, and some of the Brg-deleted cells eventually derepress CD4. These cells are blocked in differentiation and cannot exit the corresponding DN compartments, but they do not accumulate in the absence of Bcl-xL overexpression because they undergo rapid apoptosis (left panel). In contrast, Bcl-xL overexpression suppresses cell death, leading to the accumulation  of Brg-deleted cells. This is because Brg-deleted cells, derived from blood stem cells, keep feeding into DN compartments but there is no outlet, and thus these cells accumulate and fill the compartment to the rim.

 

Supplemental Figure S3. RT-PCR Analysis of Myc and NPM Expression
 

Standard curves, generated by 5-fold serial dilutions of an aRNA sample pre-amplified from total RNA isolated from WT DN4 cells (top panel), were used to quantify the abundance of Myc and NPM messages in the CD4^- CD4 cells from WT, Bclx^+/-, Bclx^+/+, Brg^F/-;Cre;Bclx^+/-, and Brg^F/–;Cre;Bclx^+/+ mice (middle panel). The PCR were done in duplicates, but only one reaction was shown in the middle panel for each sample for clarity. The high PCR efficiency and correlation coefficiency (bottom panel) testified to the quality of the reactions, and gel electrophoresis revealed a single PCR product of expected size for each reaction (not shown). 

 

Supplemental Figure S4. PCR Quantifying Abundance of Immunoprecipitated Chromatin Fragments Containing the TCF-1 or a Downstream Reference Site

 

Standard curves, generated by 3-fold dilutions of DNA precipitated with the J1 anti-Brg antibody (top panel), were used to determine the relative abundance of the TCF-1 and the reference sites precipitated by the preimmune serum (middle and bottom panels). The reactions were performed in triplicates. In this experiment, the TCF-1 and reference site precipitated by preimmune serum were 37% and 87% of that precipitated by the J1 antibody, respectively, revealing 2.4x enrichment of the TCF-1 signal.

 

Supplemental Figure S5.

 

A potential FUSE element in the mouse Myc locus upstream of the proximal TCF site (blue). The bottom panel compares the putative mouse FUSE with the human FUSE. Note the highly conserved 3¢ region in the two sequences.