Leucine Zippers

1. Leucine Zippers FRCPath 10th December 2009

2. Three families of leucine zipper proteins are known the basic region, leucine zipper proteins (bZip) the basic region, helix-loop-helix leucine zipper proteins (bHLH-Zip) homeobox DNA binding domain leucine zipper proteins leucine zipper proteins are found only in eukaryotes bZIP and bHLH-ZIP proteins found in both plants and animals homeobox leucine zipper proteins are exclusive to plants They are enhancer-type transcription factors

3. Structure of the bZIP transcription factors leucine zipper motif consists of a leucine residue 7 amino acids apart, repeating at least 3 times forms amphipathic α-helix – hydrophobic amino acids, including leucine face one direction, polar amino acids face opposite direction long side chain of leucine residue interacts with those from another polypeptide, forming the leucine zipper and resulting in dimerisation of the polypeptides the 2 α-helices are orientated in the same direction and coil around each other to form a coiled-coil motif the regular spacing of hydrophobic amino acids is critical for coiled-coil formation Beyond this region the two α-helices separate, so that the overall dimer is a Y-shaped structure leucine zipper is not DNA-binding domain region rich in basic amino acids interacts with major groove binding directly to DNA, in a sequence specific manner, is found adjacent and N-terminal to the leucine zipper motif

4. the leucine zipper facilitates dimerisation this in turn results in the correct positioning of the two basic DNA-binding domains for DNA binding once bound to DNA at a specific recognition site, the adjacent basic regions undergo a conformational change, forming an α-helix this results in a clamp or scissors-like grip around the DNA a transactivation domain is also present in the polypeptide which is thought to enhance transcription by interacting with basal transcription factors or co-activators assisting in the formation of the transcription complex on the promoter of the target gene

5. one repeating unit of a leucine zipper is referred to as a heptad the positions within the heptad are designated a, b, c, d, e, f and g the heptad begins with position g and is followed by positions a – f positions a and d reside in the hydrophobic dimerisation interface, and amino acid positions b, c and f reside on the hydrophilic face the e and g positions flank the hydrophobic face of the polypeptide in humans, d position is almost invariantly a leucine a methionine may be present in one of the heptads at position d, e.g. Myc TF position a amino acid much more variable - responsible for determining whether a leucine zipper will prefer homo- or heterodimerisation key residues affecting dimerisation are lysine and asparagine asparagine favours homodimer formation and a lysine favours heterodimer formation not due to electrostatic affinity but because the particular pairing is less unfavourable

6. charged amino acids at the g and e positions contribute to leucine zipper stability and regulate dimerisation specificity oppositely charged amino acids in the g and e’ position lie across the hydrophobic interface and interact interhelically stabilising the structure also help to regulate the specificity of bZIP protein dimerisation.

7. unique pairings of bZIP factors often result in unique pairings of DNA-binding preferences and transactivation domains bZIP proteins grouped into families based on leucine zipper similarity binding to a specific DNA sequence in a promoter is dependent on the bZIP transcription factor, homo- or heterodimerisation, and if the latter which transcription factor it has dimerised with some bZIP factors only form homodimers and some only form heterodimers, with others forming both the larger the array of bZIP factors in a genome, the greater the potential for complex transcriptional programs affecting the unique functions of individual cells, tissues and organs human genome contains 56 genes encoding proteins with bZIP motifs. experimental and predictive modelling data has calculated that ~340 unique bZip dimers probably occur leading to a complex regulation of gene expression DNA recognition sites for bZIP transcription factors have been identified in their own promoters, providing a form of autoregulation some can also be activated by phosphorylation, via both the cAMP/protein kinase A and the phosphoinositol/protein kinase C pathways

8. Structure of the bHLH-ZIP transcription factors helix-loop-helix (HLH) motif N-terminal to leucine zipper motif HLH consists of two α-helices, one short and one long, connected by a flexible loop this permits folding so that the two helices lie parallel to each other the leucine zipper motif and HLH motif mediate both DNA binding and protein dimer formation as with leucine zipper proteins a basic, DNA-binding region is present N-terminal to the HLH motif transactivaiton domains are also present, which can be N- or C-terminal to the dimerisation motifs both homo- and heterodimer formation occurs bHLH-ZIP proteins also act as enhancer-type transcription factors.

9. Function of bZIP and bHLH-ZIP transcription factors involved in many processes that are critical to the function of an organism during embryogenesis these factors are necessary for the proper development of organs and tissues such as the liver, bone and heart in adults they are involved in processes such as metabolism, circadian rhythm, and learning and memory they are also involved in cell proliferation, differentiation, survival and apoptosis have also been shown to initiate cellular responses to UV damage and osmotic stress

10. CCAAT/enhancer binding protein family (C/EBP) (bZIP) C/EBPs - 6 family members C/EBPα, β, γ, δ, ε, and ζ share substantial sequence identity (>90%) - C-terminal 55-65 amino acid residues - bZIP domain can form heterodimers in all intrafamilial combinations except for C/EBPζ interact with an identical recognition sequence, (A/G)TTGCG(C/T)AA(C/T) C/EBPζ contains two proline residues in the basic region - disrupts α-helical structure heterodimers containing this protein bind to the DNA sequence PuPuPuTGCAAT(A/C)CCC, where Pu is a purine, in the promoter regions of a subset of genes under conditions of cellular stress, to activate gene expression. C/EBPζ acts both as an inhibitor of C/EBP function and as a direct activator of other genes, depending on the cellular state. sequence similarity at the N-terminal region is low (<20%) except for three short subregions that are conserved in most members - activation domains C/EBPγ lacks an activation domain - represses gene transcription by forming inactive heterodimers

11. C/EBP proteins present in a tissue or cell type may be higher than expected from combination of homo- or heterodimerisation different sized polypeptides can be produced for C/EBPα and β either by alternative use of translation initiation codons or regulated proteolysis for C/EBPε via alternative use of promoters and differential splicing. the different isoforms have different activation potentials some only contain the leucine zipper region and act as dominant-negative inhibitors of C/EBP function by forming non-functional heterodimers as the activation potentials differs for the different isoforms, the effect on the regulation of target genes is likely to be profound. C/EBP proteins can also form heterdimers with other bZIP and non-bZIP transcription factors, adding further to the complexity of gene regulation C/EBP proteins play a pivotal role in a number of processes including cellular proliferation and differentiation, the inflammatory response, liver regeneration, metabolism, cell survival and/or apoptosis and numerous other cellular responses

12. mutations in C/EBPε have been found in patients with neutrophil-specific granule deficiency (SGD). rare congenital disorder (5 worldwide) - may be AR inheritance atypical bi-lobed nuclei and loss of expression of secondary and tertiary proteins neutrophils are defective in chemotaxis, dissaggregation, receptor up-regulation and bacterial activity Due to the numerous deficiencies and functional defects, SGD patients are severely immunocompromised develop frequent bacterial infections, including Pseudomomas aeruginosa and Staphylococcus aureas. SGD thought to involve a mutation of a myeloid-specific transcription factor C/EBPε - candidate gene - expressed primarily during granulocytic differentiation targeted disruption of the gene in mice leads to phenotypic and function defects of neutrophils, similar to patients with SGD mice are susceptible to bacterial infection and succumb to systemic infection at 3 – 5 months of age screening for mutations in two of the five individuals with SGD two different homozygous frameshift mutations found leucine zipper and DNA-binding domains lost - truncated proteins unable to activate transcription efficiently

13. heterozygous mutation identified in third patient present in basic region elevated levels of C/EBPε in the peripheral blood neutrophils of this patient markedly reduced expression of the transcription factor growth factor independence-1 (Gfi-1) which is a transcriptional repression target of C/EBPε. Gfi-1 knock-out mice have similar defects to C/EBPε knock-out mice thought overexpression of C/EBPε may contribute to disease by repressing expression of Gfi-1 no mutations in Gfi-1 found

14. five C/EBP proteins implicated in acute myeloid leukaemia (AML) and B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) Mutations of C/EBPα in a subset of sporadic and familial AML patients with normal karyotypes mutations found in N-terminal region of the protein - selective translation of a shorter protein product that lacks the ability to induce myeloid differentiation or within the bZIP domains recurrent chromosomal translocations involving the immunoglobulin heavy-chain locus (IGH) described in BCP-ALL patients fusion partner in the t(8;14) translocation - C/EBPδ more commonly C/EBPα and occasionally C/EBPγ - t(14;19) translocation ~70Kb apart at 19q13 also translocations between IGH and C/EBPβ and C/EBPε breakpoints mostly located within the 3’UTR or immediately adjacent to the C/EBP genes intact gene no mutations were found in the C/EBP fusions, exception - missense mutation in 1 C/EBPε/IGH fusion overexpression C/EBP genes detected suggests oncogenic role for the CEBP gene family in pathogenesis of BCP-ALL, although the mechanism remains to be elucidated.

15. The MiT transcription factor family (bHLH-ZIP) MiT TF family composed of 4 genes: MITF, TFEB, TFEC and TFE3 all contain bHLH-Zip motifs basic domain is ~15 amino acids in length - nearly perfectly conserved across MiT factor members interact with same DNA recognition site - E box - consensus CA(C/T)GTG form dimers with self or other family members bHLH-Zip structure and DNA binding specificity shared with members of the MYC transcription family, although they are unable to dimerise with them MITF (microphthalmia-associated transcription factor) - pivotal role in survival and differentiation of melanotcytes, necessary for neuroblast development from the neural crest, regulates genes essential for melanocytic proliferation or viability in humans, at least 4 MITF isoforms with different N-termini known existence of 4 alternative promoters - specific expression pattern of the isoforms melanocyte-specific promoter controls melanocyte-restricted expression - MITF-M isoform several potential cis-acting sites in promoter including a cAMP-responsive element involved in the regulation of MITF expression by cAMP-elevating ligands e.g. α-melanocyte stimulating hormone (αMSH). Wnt proteins also shown to play a key role in the differentiation of neural crest precursors to melanocytes.

16. mutations in MITF cause Waardenburg Syndrome Type 2A (WS2A) WS - hereditary disorder - symptoms include pigmentary abnormalities of the skin, eye and hair, congenital deafness, with or without associated face, limb and colon anomalies hearing impairment due to lack of melanocytes in the cochlea Clinically WS is divided into 4 types, WS1-4, depending on the presence or absence of symptoms other than deafness and hypopigmentation. WS1 – also have eye abnormalities WS2 – deafness and hypopigmentation only WS3 – musculoskeletal abnormalities WS4 – Hirschsprung disease WS1-3 are inherited in an autosomal dominant manner and WS4 is an autosomal recessive disorder WS1 and 3 are caused by mutations in the PAX3 gene and WS4 is caused by mutations in the SOX10 gene

17. MITF mutations - ~10-15% of WS2 cases - missense, nonsense, splicing mutations, whole and partial gene deletions Mutations result in the inability of the mutant protein to bind DNA, loss of the basic DNA-binding domain or loss of the HLH and/or leucine zipper region, so preventing dimerisation and subsequent DNA binding. loss-of-function mutations - not dominant-negative effect - mutant MITF proteins do not interfere with the DNA-binding activity of wild-type protein thought that haploinsufficiency is the cause of WS2A. one mutation C terminal to the HLH-Zip domain predicts the substitution of a serine at position 298 and was found to be a phosphorylation site Glycogen synthase kinase 3 (GSK3) has been shown to phosphorylate S298 in vitro replacement of S298 with either an alanine or proline (the latter being found in a WS2 family), disables phosphorylation of MITF by GSK3 and impairs MITF function

18. PAX3 is a paired homeodomain transcription factor - mutations cause WS1 and 3 PAX3 binds and activates the MITF promoter - regulating MITF expression SOX10 mutations are found in a proportion of WS4 sex-determining gene (SRY)-related transcription factor – binds and activates the MITF promoter Sox10 mutants fail to stimulate the MITF promoter lack of expression of MITF in WS1, 3 and 4 patients may account for the pigmentary abnormalities and deafness in these patients mutations in other genes also cause WS, e.g. endothelin B receptor, and endothelin 3 cause WS4 whether these proteins are also involved in MITF expression remains to be elucidated

19. Members of MiT family involved in acquired cancers MITF amplification found in melanoma MITF dysregulation present in clear cell sarcoma recurrent translocations of TFE3 and TFEB found in paediatric renal carcinomas and alveolar soft part sarcoma. Amplification of the 3p13-3p14 locus - location of MITF gene - found in ~20% of primary melanomas increase in MITF expression in immortalised primary human melanocytes results in a transformed phenotype - suggests that MITF functions as a melanoma oncogene in some cases Clear cell sarcoma is a high grade soft tissue sarcoma that typically arises in the tendons, aponeuroses and fascial structures of the extremities of adolescents and young adults characterised by regional and distant metastases that are poorly responsive to conventional chemotherapy or radiation therapy characterised histologically by elements of melanocytic differentiation and cytogenetically by a chromosomal translocation that fuses the Ewing’s sarcoma gene (EWS) with the CREB transcription factor family member ATF1 (bZIP TF) gene fusion results in a constitutively active transcription factor due to replacement of ATF1 regulatory phosphorylation site by the EWS domain EWS-ATF1 dysregulates MITF expression - directly binds to cAMP response element the melanocyte-specific MITF promoter MITF activation has been shown to be critical for both pigmented phenotype and survival of clear cell sarcoma

20. Translocations of MiT family members, TFE3 and TFEB found in tumours not known to share similarities to melanoma or clear cell sarcoma cytogenetic studies of paediatric renal cell carcinomas - frequent translocations in region of X chromosome where TFE3 is located (Xp11.22) TFE3 fused with several genes in renal cell carcinomas NONO at Xp13, SFPQ at 1p34, PRCC at 1q21 and CLTC at 17q23 The 3’ exons of TFE3 fuse with the 5’ exons of these genes - bHLH-Zip and transcriptional activation domains retained TFE3 translocated to the ASPL locus is also found in primary renal carcinomas and alveolar soft part sarcoma function of the amino-terminal partners of these fusion genes uncertain but all are ubiquitously expressed TFE3 translocations are found in ~30-50% of paediatric renal cell carcinomas

21. translocation between TFEB (6p21.1) and the Alpha gene (11q13) also identified in renal cell carcinomas translocation is unique, most occur in introns and the partner-derived splice donor and acceptor sites used to remove intronic sequence message derived from Alpha-TFEB translocations results in fusion of Alpha message with retained TFEB intron 1 sequence absence of splicing in keeping with Alpha gene - encodes a message that is not spliced nor has an open reading frame of significant length coding sequence of TFEB starts in exon 2, fusion results in full-length TFEB regulated by the Alpha promoter TFEB is aberrantly regulated and significantly overexpressed. multiple translocations involving TFE3 that preserve the DNA binding and transactivation domain and the unique TFEB translocation that directs expression of unaltered TFEB, suggest oncogenic activation of these MiT genes is primarily mediated through transcriptional dsyregulation MiT transcription factors bind to the same DNA sequence as MYC which is translocated into the immunoglobulin μ heavy chain enhancer in Burkitt’s lymphoma and MYCN is amplified in neuroblastoma suggests that there may be common transcriptional targets during oncogenesis

22. References Akasaka, et al. (2007) Blood109:3451-3461 Davis and Fisher (2007) Cell Cycle6:1724-1729 Gombart, et al. (2001) Blood 97:2561-2567 Khanna-Gupta, et al. (2007) Blood109:4181-4190 Nobukuni, et al. (1996) Am J Hum Genet59:76-83 Ramji and Foka (2002) Biochem J 365:561-575 Takeda, et al. (2000) Hum Mol Genet9:125-132 Tassabehji, et al. (1994) Nat Genet8:251-255 Verastegui, et al. (2000) J Biol Chem 275:30757-30760