Research projects

The main aim of this project is to characterise the role of the acetylation of Lys16 of the H4 in neural differentiation. To this end it is planned to study its levels in general and its nuclear distribution, determining total acetylomes in connection with the degree of cellular differentiation, its genetic expression profiles and the degree of chromatin compacting in pluripotent cells and differentiated cells in different neural lines.

From the start of this project work has taken place towards two partial objectives:

1. The preparation of an in vitro neural differentiation protocol. For this, work commenced using the embryonic mouse cell line JM8A3, and a protocol was followed that basically consists of 3 parts: the growth of the cells in a matrix of embryonic mouse fibroblasts; the subsequent formation of neural precursors by means of culture in suspension with retinoic acid and, finally, differentiation into neurons in a matrix of poly-D-ornithine and laminine. In each of these points, overall acetylation in H4K16ac was analysed by means of HPCE, the results of which showed higher levels of acetylation in lysine 16 of histone H4 in the most differentiated cells.
2. The preparation of a ChiP-seq protocol to analyse the distribution of the chromatin of H4K16ac. ChIP-Seq is a highly interesting tool for the study of interactions between DNA and proteins, although it is a complex assay. For this reason, it is planned to draw up a protocol that optimises the results of ChIP-Seq in the study of H4K16ac within the experimental system. To identify the conditions of ChIP which make it possible to obtain optimum DNA fragments for use in ultrasequencing, a comparative study was made of several of the key points of the technique: the number of cells at the start, the intercrossing of DNA and histones, chromatin fragmentation, the method of DNA extraction and the antibody used in immunoprecipitation. Following preparation, analysis is now taking place of the acetylation pattern of H4K16 in pluripotent and differentiated cells. There are now fragments of immunoprecipitated DNA that will be ultrasequenced in forthcoming weeks on a Hi-Seq 2000 Illumina platform.