Structure and Function

The C-terminus of the CEBP genes is highly conserved, with a sequence similarity of >90%. This region contains the leucine zipper and basic DNA binding domains and the sequence similarity translates into similar structural and functional properties of all family members (Figure 1.9). The leucine zipper consists of a heptad of leucine repeats, which form an alpha helix (Vinson et al., 1993). This protein structure allows bZIP family members to homo and heterodimerise with each other; a crucial step in the action of the CEBPs, which stabilises the proteins interaction with target DNA (Cooper et al., 1995; Parkin et

al., 2002). The DNA binding domain is located upstream of the bZIP, the function

of which is determined by approximately 20 amino acids in the region (Johnson, 1993). The N-terminus of the CEBP proteins contain the transactivating and

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regulatory domains, which allow protein interactions. This region is more variable than the C-terminus. Different CEBP genes and isoforms express different numbers of transactivating domains (TD). These domains have been shown to exert varying potencies of transactivation and have been observed to bind different targets, but also act synergistically (Nerlov and Ziff, 1995). Differing isoform function is in part conferred by variation in which TD is expressed. For example, CEBPA contains three TDs: TDI, TDII and TDIII, with different CEBPA isoforms expressing different TDs. Isoform p42 expresses the most powerful TD, TDI, which is lacking in the shorter p30 isoform (Figure 1.9). CEBPG contains no TDs, giving rise to the theory that CEBPG is a dominant negative isoform functioning by inactivation of other CEBP and bZIP family members. The other

CEBPs contain varying numbers of TDs depending upon the gene and protein

isoform (Figure 1.9).

Regulatory domains are also present in the N-terminus, with four of the CEBP genes expressing these domains (Figure 1.9), which function in an inhibitory manner. Regulatory domain I has been shown to have binding sequences for the small ubiquitin-related modifier (SUMO) protein, which post transcriptionally modifies its targets and generally functions by SUMOylating and inhibiting transcription factors. Further research has since discovered that SUMOylation exerts a regulatory effect on specific CEBP isoforms in several different settings, exerted by multiple SUMO family genes (Eaton and Sealy, 2003; Wang et al., 2006; Khanna-Gupta, 2008).

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Figure 1.9. IGH partnered CEBP protein domain loci, with protein isoforms.

The proteins show a conserved C-terminal region with the leucine zipper domain, allowing for protein heterodimerisation, and a DNA binding domain. The N-terminus shows more variation with different combinations of transactivating and regulatory domains present, dependent upon gene and protein isoform. CEBPA is predominantly expressed as two isoforms, the full length p42 isoform and the regulatory p30 isoform. CEBPB is expressed as three isoforms, the activating LAP* and LAP isoforms, and the negative LIP isoform. CEBPD is expressed as a single isoform. CEBPE is expressed as four p32, p30, p27 and p14 isoforms whose interplay controls terminal granulocytic differentiation. CEBPG shows no activating or regulatory domains and is believed to mainly function as a dominant negative protein for the other CEBP members. Adapted from (Schrem et al., 2004).

CEBP isoforms are created by altered ribosomal translation points, which begin

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1993). This translation is controlled by the mTOR pathway, which determines

CEBP isoform ratios through the elF4E gene (Calkhoven et al., 2000).

Isoform expression is particularly important for CEBPA, CEBPB and CEBPE, all of which have dominant negative isoforms influencing the function of their full length CEBP proteins (Figure 1.9). The CEBP proteins exhibit largely identical DNA binding specificities in vitro, making it difficult to predict which CEBP protein will bind to which sequences (Tsukada et al., 2011). Although DNA binding specificity is generally unique, the CEBP proteins can function in place of each other. For example, in haematopoiesis and adipocyte differentiation, CEBPA can function in place of CEBPB (Jones et al., 2002; Chiu et al., 2004). Synergy has also been observed in CEBP heterodimers, with CEBPB and CEBPD acting in concert in inflammatory signalling (Yan et al., 2012). In contrast CEBPA and

CEBPD play opposing roles in hypoxia signalling through the HIF-1α gene, with CEBPA inhibiting its function and CEBPD supporting HIF-1α expression

(Balamurugan and Sterneck, 2013). Other examples will be discussed below. As well as forming hetero- and homodimers within the CEBP family, the proteins can also form heterodimers with other bZIP family members, such as the

CREB/ATF and FOS/JUN family, generally to facilitate existing CEBP functions,

such as immune signalling and response, as well as differentiation control (Tsukada et al., 1994; Newman and Keating, 2003). They also repress CEBP function (Podust et al., 2001).

While the CEBP genes act in concert in haematopoiesis (Figure 1.10) and in the development of other tissues, there is a level of complexity in their interplay, both in specific tissues and with each other. It is understood that the proteins can and do heterodimerize, but the results of such dimerization are largely unclear. Even known functions can be inverted depending upon context. Below is a brief description of some of the many functions of the CEBP gene family members (Figure 1.10).

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Figure 1.10. Natural and induced CEBP expression in haematopoiesis from published reports (Tsukada et al., 2011). Blue arrows indicate natural lineage progression, orange arrows indicate experimentally induced differentiation (Heavey et al., 2003; Xie et al., 2004). CEBP expression is mostly sequential, beginning with CEBPA directing haematopoiesis down the myeloid lineage, and continuing with interplay between the CEBP genes in CMPs and myeloblasts and ending with specific CEBPs contributing to the terminal differentiation of the branched myeloid lineages. CEBPB and CEBPD expressed in monocytes and macrophages, and CEBPE and CEBPA expressed in terminal granulocytes. CEBPs importance in myeloid differentiation was underlined upon induced expression in lymphocytes leading to forced differentiation into myeloid cells.