Identification of novel distal regulatory elements of the human neuroglobin gene

Abstract

Neuroglobin (Ngb), a new member of the vertebrate globin family, is localized predominantly in the neurons, retina and a number of endocrine tissues. Although the physiological function is still unclear, accumulating evidence demonstrated that Ngb is involved in cellular oxygen homeostasis and functions as a neuroprotectant. Ngb is found to protect cells and tissues from hypoxic and ischemic damage and has the ability to alleviate the symptoms of stroke and Alzheimer’s disease in rodent’s brain.

In the present study, we hypothesize that distal regulatory elements are involved in the optimal expression of the Ngb gene. The objective of this project is to search for distal regulatory elements of the human Ngb gene and to characterize the underlying mechanism for Ngb gene tissue-specific expression.

By chromosome conformation capture (3C) technique we identified two novel distal regulatory elements located -70kb upstream and +100kb downstream from the Ngb gene. ENCODE database showed the presence of DNaseI hypersensitive and multiple transcription factor binding sites in these regions. Further analyses using luciferase reporter assay and electrophoretic mobility shift assays (EMSA) suggested that the -70kb region upstream of the Ngb gene contained a neuronal-specific enhancer and the presence of GATA transcription factor binding sites. In vivo studies by chromatin immunoprecipitation (ChIP) analysis further confirmed the specific binding of GATA-2 and histone hyperacetylation at the -70kb element. Knockdown of GATA-2 caused Ngb expression level to drop dramatically, indicating GATA-2 as an essential transcription factor for the activation of Ngb expression.

Taken together, I show that the Ngb gene is regulated by cell type-specific loop formation between its promoter and a newly identified GATA-2 bound distal regulatory element. This element is a bona fide distal regulatory element for Ngb gene and may be a key regulator for the spatial and temporal expression of the Ngb gene.