HEB in the Spotlight: Transcriptional Regulation of T-Cell Specification, Commitment, and Developmental Plasticity

The development of T cells from multipotent progenitors in the thymus occurs by cascades of interactions between signaling molecules and transcription factors, resulting in the loss of alternative lineage potential and the acquisition of the T-cell functional identity. These processes require Notch signaling and the activity of GATA3, TCF1, Bcl11b, and the E-proteins HEB and E2A. We have shown that HEB factors are required to inhibit the thymic NK cell fate and that HEBAlt allows the passage of T-cell precursors from the DN to DP stage but is insufficient for suppression of the NK cell lineage choice. HEB factors are also required to enforce the death of cells that have not rearranged their TCR genes. The synergistic interactions between Notch1, HEBAlt, HEBCan, GATA3, and TCF1 are presented in a gene network model, and the influence of thymic stromal architecture on lineage choice in the thymus is discussed

Downregulation of histone H2A and H2B pathways is associated with anthracycline sensitivity in breast cancer

Abstract Background Drug resistance in breast cancer is the major obstacle to effective treatment with chemotherapy. While upregulation of multidrug resistance genes is an important component of drug resistance mechanisms in vitro, their clinical relevance remains to be determined. Therefore, identifying pathways that could be targeted in the clinic to eliminate anthracycline-resistant breast cancer remains a major challenge. Methods We generated paired native and epirubicin-resistant MDA-MB-231, MCF7, SKBR3 and ZR-75-1 epirubicin-resistant breast cancer cell lines to identify pathways contributing to anthracycline resistance. Native cell lines were exposed to increasing concentrations of epirubicin until resistant cells were generated. To identify mechanisms driving epirubicin resistance, we used a complementary approach including gene expression analyses to identify molecular pathways involved in resistance, and small-molecule inhibitors to reverse resistance. In addition, we tested its clinical relevance in a BR9601 adjuvant clinical trial. Results Characterisation of epirubicin-resistant cells revealed that they were cross-resistant to doxorubicin and SN-38 and had alterations in apoptosis and cell-cycle profiles. Gene expression analysis identified deregulation of histone H2A and H2B genes in all four cell lines. Histone deacetylase small-molecule inhibitors reversed resistance and were cytotoxic for epirubicin-resistant cell lines, confirming that histone pathways are associated with epirubicin resistance. Gene expression of a novel 18-gene histone pathway module analysis of the BR9601 adjuvant clinical trial revealed that patients with low expression of the 18-gene histone module benefited from anthracycline treatment more than those with high expression (hazard ratio 0.35, 95 % confidence interval 0.13–0.96, p = 0.042). Conclusions This study revealed a key pathway that contributes to anthracycline resistance and established model systems for investigating drug resistance in all four major breast cancer subtypes. As the histone modification can be targeted with small-molecule inhibitors, it represents a possible means of reversing clinical anthracycline resistance. Trial registration ClinicalTrials.gov identifier NCT00003012 . Registered on 1 November 1999

Regulation of Early T-cell Development and Commitment by HEB

Early T-cell development is regulated by a complex interplay between transcription factors and developmental cues which ensure that functional T-cells are produced within the thymus. Early thymocytes integrate these signals in a step-wise fashion that progressively restricts their lineage potential as they transition through the early stages of T-cell development. Gene knockout studies have shown that the E-protein transcription factor HEB is required for normal thymocyte development. Furthermore, many additional key regulators such as Notch1 have been identified, but the connections among them and their specific roles in early T-cell development have not been well established. In this thesis, I set out to determine the specific roles of HEB at the beta-selection checkpoint and to establish connections between HEB and the key regulators within the gene regulatory network that orchestrates early T-cell development. To facilitate these studies, I generated a series of new mouse models including HEBAlt transgenic mice that express a short form of HEB called HEBAlt, which enabled me to answer specific questions and examine rare populations. First, my studies of HEB-/- mice allowed me to identify an early block in T-cell development, which was alleviated upon the addition of an HEBAlt transgene. Furthermore, I identified pTa and CD3e signalling as specific targets of HEBAlt during -selection. Second, my studies on HEB-/- mice revealed that they have a defect in T-cell commitment, with compromised Notch1 function and a tendency to become DN1-like cells. Moreover, the DN1-like cells could be induced to differentiate into thymic NK cells, revealing a role for HEB in the T/NK cell lineage decision. This study has revealed a new set of interactions among HEB, Notch1, and GATA3 that regulate the T-cell fate choice in developing thymocytes. Unexpectedly, my studies have also provided evidence for a role of HEBAlt in lymphomagenesis, highlighting the strict regulation of E-protein function that is necessary to ensure normal T-cell development.Ph

The small heat shock protein HSPB5 attenuates the severity of lupus nephritis in lupus-prone mice

Lupus nephritis (LN) is a common and serious complication of systemic lupus erythematosus. The current treatments for LN are accompanied with severe immunotoxicity and have limits of effectiveness. Since our in vitro experiments demonstrated that a small heat shock protein (HSP), alpha-B crystallin (HSPB5; CRYAB), selectively modulates myeloid cells towards anti-inflammatory and tolerogenic phenotypes, the aim of this study was to investigate whether HSPB5 can attenuate the severity of LN. MRL/lpr mice were treated intravenously with HSPB5 at 2.5 or 10 μg/dose twice per week after disease onset, from 11 to 21 weeks of age. Disease progression was monitored by weekly measurements of proteinuria, and sera, spleens, and kidneys were collected for assessment at the terminal time point. Treatment with 10 μg HSPB5 substantially reduced endocapillary proliferation and tubular atrophy, which significantly reduced proteinuria and blood urea nitrogen (BUN). Compared to vehicle, 10 μg HSPB5 treatment substantially decreased activation/proliferation of splenocytes, increased IL-10+ macrophages, T and B regulatory cells (Treg, Breg), increased serum IL-10, and lowered expression of IL-6 in kidneys, which correlated with improved kidney function and pathology. This study demonstrated the utility of exogenous human HSPB5 to attenuate severe nephropathy in MRL/lpr mice and provides evidence in favour of a novel therapeutic approach for lupus nephritis

The Basic Helix-Loop-Helix Transcription Factor HEBAlt Is Expressed in Pro-T Cells and Enhances the Generation of T Cell Precursors

The basic helix-loop-helix (bHLH) transcription factors HEB and E2A are critical mediators of gene regulation during lymphocyte development. We have cloned a new transcription factor, called HEBAlt, from a pro-T cell cDNA library. HEBAlt is generated by alternative transcriptional initiation and splicing from the HEB gene locus, which also encodes the previously characterized E box protein HEBCan. HEBAlt contains a unique N-terminal coding exon (the Alt domain) that replaces the first transactivation domain of HEBCan. Downstream of the Alt domain, HEBAlt is identical to HEBCan, including the DNA binding domain. HEBAlt is induced in early thymocyte precursors and down-regulated permanently at the double negative to double positive (DP) transition, whereas HEBCan mRNA expression peaks at the DP stage of thymocyte development. HEBAlt mRNA is up-regulated synergistically by a combination of HEBCan activity and Delta-Notch signaling. Retroviral transduction of HEBAlt or HEBCan into hemopoietic stem cells followed by OP9-DL1 coculture revealed that HEBAlt-transduced precursors generated more early T lineage precursors and more DP pre-T cells than control transduced cells. By contrast, HEBCan-transduced cells that maintained high level expression of the HEBCan transgene were inhibited in expansion and progression through T cell development. HEB−/− fetal liver precursors transduced with HEBAlt were rescued from delayed T cell specification, but HEBCan-transduced HEB−/− precursors were not. Therefore, HEBAlt and HEBCan are functionally distinct transcription factors, and HEBAlt is specifically required for the efficient generation of early T cell precursors

Additional file 1: Figure S1. of Downregulation of histone H2A and H2B pathways is associated with anthracycline sensitivity in breast cancer

Clinical trial BR9601 information. (a) Schematic representation. (b) Patient information. Figure S2. Entire FI network from 61 consistently changing genes. Red circles = upregulated genes; green circles = downregulated genes; diamonds = linker genes. Figure S3. Heat maps of probes for the 61 consistently changing genes. Rows are labelled with gene symbol and microarray probe IDs. (a) Raw expression values. (b) Row scaled expression values. Figure S4. Combination of pre-processing methods. The most optimal method selected was at the top(high-rank). Figure S5. Sample-by-gene heat map. Rows represent patients and columns represent genes. Both were clustered using a ward clustering algorithm. Figure S6. FI network generated from the histone module. Circles = genes within the module, diamonds = linker genes. Figure S7. Multiplot showing scaled mRNA abundance levels for each gene. A treatment-by-marker interaction Cox proportional hazards model was fit for each gene, and results are visualized on the right with the squares representing the hazard ratios (HRs) and the ends of the segments representing the 95 % confidence intervals in log2 scale. Patients are sorted by DRFS events on the x-axis and genes by decreasing log2 HR on the y-axis. Figure S8. Flow cytometric analysis of HER2 in MCF7 cells. Yellow dotted line represents HER2- cell line (MDA-MB-231), while purple dotted line shows HER2 amplified cell line (SKBR3). Green and blue solid lines represent native and epirubicin-resistant MCF7 cell lines, respectively, and show they are negative for HER2 cell surface expression. Table S1. Doubling times (in hours) of breast cancer cell lines. Table S2 List of 61 common genes with consistent differential expression across all four cell lines. Table S3. Percentage reduction in gene expression compared with non-targeting siRNA control. Table S4. Drugs targeting epirubicin-resistant breast cancer cells. Table S5. List of primary antibodies. Table S6. List of histone module genes in the NanoString CodeSet. (PDF 1653 kb

Additional file 2: of Downregulation of histone H2A and H2B pathways is associated with anthracycline sensitivity in breast cancer

Description of mRNA abundance data processing, module dysregulation score (MDS) and survival modelling. (PDF 360 kb