State-of-the-art and preparatory work

It is becoming increasingly evident that direct interactions between promoters and distant regulatory elements play a key role in regulation of eukaryotic gene expression (1, 2). Assembly of multiple regulatory elements into a single complex (chromatin hub) is an essential step of activation of tissue specific gene expression. It is not quite clear how stable the above‐mentioned chromatin hubs are and what kind of interactions support their integrity. One supposition is that mutual interactions of transcription factors bound to distant regulatory elements are sufficient to hold these elements in a single complex. The other possibility is that interactions between distant regulatory elements are supported by the nuclear matrix or by special proteins which can be referred to as “communicator proteins”, e.g.: Su(Hw) (3) and CTCF (4). Although the conception of chromatin hubs was developed as an alternative to the flip‐flop hypothesis (5) of activation of multiple genes by a single enhancer and chromatin hubs were considered static rather than dynamic (2), our recent data question this point of view (6). The aim of the present project is to extend our knowledge about the threedimensional organization of the eukaryotic genome and, in particular, about the role of this organization in regulation of gene expression. To this end it is important to mention that the conception of chromatin hubs is presently based on the results of studying a few genomic models. It seems thus important to characterise other genomic domains. In the frame of the present project we are planning to characterize the spatial organization of the human AML gene domain in erythroid and lymphoid cells. Besides contribution to fundamental knowledge the results of this work may be of practical importance because the AML1 gene is frequently involved in chromosomal translocations (7). Simultaneously we are planning to characterize the spatial organization of other genomic domains including the c‐myc gene domain. Previous work of our lab has demonstrated that a DNA loop anchorage region (LAR) is located in the upstream flanking area of the human c‐myc gene (8). It will be interesting to find out how this LAR is situated in respect to CTCF binding sites and whether these CTCF binding sites are essential for DNA anchorage on the nuclear matrix.

References

1. Patrinos, G. P., de Krom, M., de Boer, E., Langeveld, A., Imam, A. M., Strouboulis, J., de Laat, W. & Grosveld, F. G. (2004) Multiple interactions between regulatory regions are required to stabilize an active chromatin hub. Genes Dev 18, 1495‐ 1509.

2. de Laat, W. & Grosveld, F. (2003) Spatial organization of gene expression: the active chromatin hub. Chromosome Res. 11, 447‐459.

3. Muravyova, E., Golovnin, A., Gracheva, E., Parshikov, A., Belenkaya, T., Pirrotta, V. & Georgiev, P. (2001) Loss of insulator activity by paired Su(Hw) chromatin insulators. Science 291, 495‐498.

4. Splinter, E., Heath, H., Kooren, J., Palstra, R. J., Klous, P., Grosveld, F., Galjart, N. & de Laat, W. (2006) CTCF mediates long‐range chromatin looping and local histone modification in the beta‐globin locus. Genes Dev 20, 2349‐2354.

5. Wijgerde, M., Grosveld, F. & Fraser, P. (1995) Transcription complex stability and chromatin dynamics in vivo. Nature 377, 209‐213.

6. Philonenko, E. S., Klochkov, D. B., Borunova, V. V., Gavrilov, A. A., Razin, S. V. & Iarovaia, O. V. (2009) TMEM8 ‐ a nonglobin gene entrapped in the globin web. Nucleic Acids Res 37, 7394‐7406.

7. Bystritskiy, A. A. & Razin, S. V. (2004) Breakpoint clusters: reason or consequence? Crit Rev Eukaryot Gene Expr 14, 65‐ 78.

8. Gromova, II, Thomsen, B. & Razin, S. V. (1995) Different topoisomerase II antitumor drugs direct similar specific longrange fragmentation of an amplified c‐MYC gene locus in living cells and in high‐salt extracted nuclei. Proc Natl Acad Sci U S A 92, 102‐106.

Aims

The overall aim of this project is to disclose the basic principles of organization of tissue‐specific active chromatin hubs. In detail, the aims of this project are

I. to characterize the spatial organization of the AML1 gene in lymphoid and erythroid cells

II. to find out if an assembly of short‐lived alternative active chromatin hubs is a general phenomenon in eukaryotic cells

III. to study the possible role of CTCF in formation of active and repressive chromatin loops.

Work programme and methods

The main methods to be used in the project are (i) chromosome conformation capture (3C), (ii) chromatin immunoprecipitation (ChIP), (iii) functional assays based on transient expression of reporter genes in eukaryotic cells, (iv) FISH, immunostaining and confocal microscopy.

Titles for dissertations (prospective)

• Characterization of regulatory elements which control expression of human AML1 gene in erythroid and lymphoid cells

• Mapping of transcription factories in chicken chromosome 14

• Visualisation of clusters of CpG islands in the nucleus

• The role of the nucleolus in DNA repair

Relationships/connections within the research training group

Renkawitz: Chromatin remodelling directed by the nuclear lamina

Benefits of the scientific exchange

The laboratories of S.V. Razin and R. Renkawitz (Giessen) work in the same area of research and in many particular fields have complementary methodological experience. This justifies the importance of the collaboration.