Altered chromatin occupancy of master regulators underlies evolutionary divergence in the transcriptional landscape of erythroid differentiation.

Erythropoiesis is one of the best understood examples of cellular differentiation. Morphologically, erythroid differentiation proceeds in a nearly identical fashion between humans and mice, but recent evidence has shown that networks of gene expression governing this process are divergent between species. We undertook a systematic comparative analysis of six histone modifications and four transcriptional master regulators in primary proerythroblasts and erythroid cell lines to better understand the underlying basis of these transcriptional differences. Our analyses suggest that while chromatin structure across orthologous promoters is strongly conserved, subtle differences are associated with transcriptional divergence between species. Many transcription factor (TF) occupancy sites were poorly conserved across species (∼25% for GATA1, TAL1, and NFE2) but were more conserved between proerythroblasts and cell lines derived from the same species. We found that certain cis-regulatory modules co-occupied by GATA1, TAL1, and KLF1 are under strict evolutionary constraint and localize to genes necessary for erythroid cell identity. More generally, we show that conserved TF occupancy sites are indicative of active regulatory regions and strong gene expression that is sustained during maturation. Our results suggest that evolutionary turnover of TF binding sites associates with changes in the underlying chromatin structure, driving transcriptional divergence. We provide examples of how this framework can be applied to understand epigenomic variation in specific regulatory regions, such as the ß-globin gene locus. Our findings have important implications for understanding epigenomic changes that mediate variation in cellular differentiation across species, while also providing a valuable resource for studies of hematopoiesis.

Exome sequencing results in successful diagnosis and treatment of a severe congenital anemia.

Whole-exome sequencing is increasingly used for diagnosis and identification of appropriate therapies in patients. Here, we present the case of a 3-yr-old male with a lifelong and severe transfusion-dependent anemia of unclear etiology, despite an extensive clinical workup. Given the difficulty of making the diagnosis and the potential side effects from performing interventions in patients with a congenital anemia of unknown etiology, we opted to perform whole-exome sequencing on the patient and his parents. This resulted in the identification of homozygous loss-of-function mutations in the EPB41 gene, encoding erythrocyte protein band 4.1, which therefore causes a rare and severe form of hereditary elliptocytosis in the patient. Based on prior clinical experience in similar patients, a surgical splenectomy was performed that resulted in subsequent transfusion independence in the patient. This case illustrates how whole-exome sequencing can lead to accurate diagnoses (and exclusion of diagnoses where interventions, such as splenectomy, would be contraindicated), thereby resulting in appropriate and successful therapeutic intervention-a major goal of precision medicine.

A primer on the current state of microarray technologies.

DNA microarray technology has been used for genome-wide gene expression studies that incorporate molecular genetics and computer science analyses on massive levels. The availability of microarrays permit the simultaneous analysis of tens of thousands of genes for the purposes of gene discovery, disease diagnosis, improved drug development, and therapeutics tailored to specific disease processes. In this chapter, we provide an overview on the current state of common microarray technologies and platforms. Since many genes contribute to normal functioning, research efforts are moving from the search for a disease-specific gene to the understanding of the biochemical and molecular functioning of a variety of genes whose disrupted interaction in complicated networks can lead to a disease state. The field of microarrays has evolved over the past decade and is now standardized with a high level of quality control, while providing a relatively inexpensive and reliable alternative to studying various aspects of gene expression.