Erythroid lineage chromatin accessibility maps facilitate identification and validation of NFIX as a fetal hemoglobin repressor.

Human genetics has validated de-repression of fetal gamma globin (HBG) in adult erythroblasts as a powerful therapeutic paradigm in diseases involving defective adult beta globin (HBB). To identify factors involved in the switch from HBG to HBB expression, we performed Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) on sorted erythroid lineage cells derived from bone marrow (BM) or cord blood (CB), representing adult and fetal states, respectively. BM to CB cell ATAC-seq profile comparisons revealed genome-wide enrichment of NFI DNA binding motifs and increased NFIX promoter chromatin accessibility, suggesting that NFIX may repress HBG.

Selective Inhibitors of Histone Deacetylases 1 and 2 Synergize with Azacitidine in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a heterogeneous group of hematopoietic stem cell disorders characterized by defects in myeloid differentiation and increased proliferation of neoplastic hematopoietic precursor cells. Outcomes for patients with AML remain poor, highlighting the need for novel treatment options. Aberrant epigenetic regulation plays an important role in the pathogenesis of AML, and inhibitors of DNA methyltransferase or histone deacetylase (HDAC) enzymes have exhibited activity in preclinical AML models.

Chemical Inhibition of Histone Deacetylases 1 and 2 Induces Fetal Hemoglobin through Activation of GATA2.

Therapeutic intervention aimed at reactivation of fetal hemoglobin protein (HbF) is a promising approach for ameliorating sickle cell disease (SCD) and β-thalassemia. Previous studies showed genetic knockdown of histone deacetylase (HDAC) 1 or 2 is sufficient to induce HbF. Here we show that ACY-957, a selective chemical inhibitor of HDAC1 and 2 (HDAC1/2), elicits a dose and time dependent induction of γ-globin mRNA (HBG) and HbF in cultured primary cells derived from healthy individuals and sickle cell patients.

HDAC1,2 inhibition impairs EZH2- and BBAP-mediated DNA repair to overcome chemoresistance in EZH2 gain-of-function mutant diffuse large B-cell lymphoma.

Gain-of-function mutations in the catalytic site of EZH2 (Enhancer of Zeste Homologue 2), is observed in about 22% of diffuse large B-cell lymphoma (DLBCL) cases. Here we show that selective inhibition of histone deacetylase 1,2 (HDAC1,2) activity using a small molecule inhibitor causes cytotoxic or cytostatic effects in EZH2 gain-of-function mutant (EZH2GOF) DLBCL cells. Our results show that blocking the activity of HDAC1,2 increases global H3K27ac without causing a concomitant global decrease in H3K27me3 levels.

Effects of β4 integrin expression on microRNA patterns in breast cancer.

The integrin α6β4 is defined as an adhesion receptor for laminins. Referred to as 'β4', this integrin plays a key role in the progression of various carcinomas through its ability to orchestrate key signal transduction events and promote cell motility. To identify novel downstream effectors of β4 function in breast cancer, microRNAs (miRNAs) were examined because of their extensive links to tumorigenesis and their ability to regulate gene expression globally.

Integrin β4 regulates SPARC protein to promote invasion.

The α6β4 integrin (referred to as "β4" integrin) is a receptor for laminins that promotes carcinoma invasion through its ability to regulate key signaling pathways and cytoskeletal dynamics. An analysis of published Affymetrix GeneChip data to detect downstream effectors involved in β4-mediated invasion of breast carcinoma cells identified SPARC, or secreted protein acidic and rich in cysteine. This glycoprotein has been shown to play an important role in matrix remodeling and invasion.

Global DNA demethylation during mouse erythropoiesis in vivo.

In the mammalian genome, 5'-CpG-3' dinucleotides are frequently methylated, correlating with transcriptional silencing. Genome-wide demethylation is thought to occur only twice during development, in primordial germ cells and in the pre-implantation embryo. These demethylation events are followed by de novo methylation, setting up a pattern inherited throughout development and modified only at tissue-specific loci.

Identification and analysis of mouse erythroid progenitors using the CD71/TER119 flow-cytometric assay.

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of red cell formation is regulated by the hormone erythropoietin (Epo), whose synthesis is triggered by tissue hypoxia. A threat to adequate tissue oxygenation results in a rapid increase in Epo, driving an increase in erythropoietic rate, a process known as the erythropoietic stress response.

A key commitment step in erythropoiesis is synchronized with the cell cycle clock through mutual inhibition between PU.1 and S-phase progression.

Hematopoietic progenitors undergo differentiation while navigating several cell division cycles, but it is unknown whether these two processes are coupled. We addressed this question by studying erythropoiesis in mouse fetal liver in vivo. We found that the initial upregulation of cell surface CD71 identifies developmentally matched erythroblasts that are tightly synchronized in S-phase.

TWEAK/Fn14 pathway: a nonredundant role in intestinal damage in mice through a TWEAK/intestinal epithelial cell axis.

Background And Aims: Tumor necrosis factor (TNF) superfamily members have attracted attention as new therapeutic targets for treating inflammatory disease. TNF-like weak inducer of apoptosis (TWEAK) is a unique, multifunctional TNF family cytokine that signals through its receptor, fibroblast growth factor-inducible molecule 14 (Fn14). The role of this pathway in the intestine has not been previously reported.

Increased levels of transforming growth factor beta receptor type I and up-regulation of matrix gene program: A model of scleroderma.

Objective: Previously published studies have demonstrated that a majority of systemic sclerosis (SSc) fibroblasts exhibit elevated levels of transforming growth factor beta type I receptor (TGFbetaRI). An experimental model that recapitulates this condition was established in control dermal fibroblasts by titrating the dose of adenovirus vector expressing TGFbetaRI (AdTGFbetaRI). The present study was undertaken to determine the functional consequences of increased levels of TGFbetaRI in SSc.

A sequence-oriented comparison of gene expression measurements across different hybridization-based technologies.

Over the last decade, gene expression microarrays have had a profound impact on biomedical research. The diversity of platforms and analytical methods available to researchers have made the comparison of data from multiple platforms challenging. In this study, we describe a framework for comparisons across platforms and laboratories.

Gene profiling of scleroderma skin reveals robust signatures of disease that are imperfectly reflected in the transcript profiles of explanted fibroblasts.

Objective: To determine whether biopsy specimens obtained from systemic sclerosis (SSc) lesions show a distinctive gene profile, whether that gene profile is maintained in fibroblasts cultured from SSc skin biopsy specimens, and whether results from tissue obtained from multiple clinical centers can be combined to yield useful observations in this rare disease.

Methods: Biopsy samples and passaged fibroblasts were stored in RNAlater solution prior to processing for RNA. RNA from SSc and control skin biopsy specimens, as well as SSc and control explanted passage 4 fibroblasts, from 9 patients and 9 controls was hybridized to Affymetrix HG-U133A arrays.

Accurate and precise transcriptional profiles from 50 pg of total RNA or 100 flow-sorted primary lymphocytes.

We have developed a total RNA amplification and labeling strategy for use with Affymetrix GeneChips. Our protocol, which we denote BIIB, employs two rounds of linear T7 amplification followed by Klenow labeling to generate a biotinylated cDNA. In benchmarking studies using a titration of mouse universal total RNA, BIIB outperformed commercially available kits in terms of sensitivity, accuracy, and amplified target length, while providing equivalent results for technical reproducibility.

Application of functional genomic technologies in a mouse model of retinal degeneration.

Generation of tissue-specific, normalized and subtracted cDNA libraries has the potential to characterize the expression of rare transcriptional units not represented on Affymetrix GeneChips. Initial sequence analysis of our murine cDNA clone collections showed that as much as 86, 45, and 30% of clones are not represented on the Affymetrix Mu11k, MG-U74, and MG-430 chip sets, respectively. A detailed study that compared EST sequences of a subtracted library generated from mouse retina to those of MG-430 consensus sequences was undertaken, using UniGene build 124 as the common reference.

Nondestructive quality control for microarray production.

The use of microarrays to monitor gene expression has become a standard research tool at both academic and industrial research institutions. Quality control of common printing defects during DNA deposition onto glass substrates is critical to maintaining data integrity and preventing the needless consumption of precious RNA, labeling reagents, and time. Here we demonstrate a nondestructive method for monitoring the quality of every spot on every chip of a microarray production run.