Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Gene Section Review

HRK (harakiri, BCL2 interacting (contains only BH3 domain)) Jonathan Ham Molecular Haematology and Cancer Biology Unit, UCL Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK (JH)

Published in Atlas Database: July 2011 Online updated version : http://AtlasGeneticsOncology.org/Genes/HRKID40865ch12q24.html DOI: 10.4267/2042/46085 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity following Nerve Growth Factor (NGF) withdrawal in sympathetic neurons, neuronally differentiated PC12 Other names: DP5; HARAKIRI; BID3 cells and dorsal root ganglion (DRG) neurons, KCl HGNC (Hugo): HRK deprivation in cerebellar granule neurons, beta amyloid treatment in cortical neurons and following axotomy of Location: 12q24.22 motor neurons in vivo (Imaizumi et al., 1997; Imaizumi Local order: According to Ensembl and the NCBI et al., 1999; Harris and Johnson, 2001; Imaizumi et al., Map Viewer, the flanking HRK in the plus strand 2004; Coultas et al., 2007; Ma et al., 2007; Towers et direction are: RNFT2, which is downstream of HRK al., 2009). Experiments with HRK-/- knockout mice and transcribed towards it, and FBXW8, which is have shown that HRK is not essential for normal upstream of HRK and transcribed divergently. embryonic development but does contribute to the Note: HRK / Dp5 / Bid3 is a BH3-only member of the death of DRG neurons following NGF deprivation in Bcl-2 family of regulators. Bcl-2 family vitro (Coultas et al., 2007) and the death of regulate the mitochondrial (intrinsic) pathway motoneurons in vivo following hypoglossal nerve of apoptosis by regulating mitochondrial outer transection (Imaizumi et al., 2004). membrane permeability (Chipuk et al., 2010). Interactions between the pro- and anti-apoptotic DNA/RNA members of the Bcl-2 family determine the fate of cells in response to signals that induce apoptosis. BH3-only Description proteins are activated in response to a variety of signals The human HRK spans 20206 bases, telomere to including survival factor withdrawal, DNA damage, ER centromere orientation. Exon 2, which encodes the 3 stress and oxidative stress. HRK expression increases UTR of the HRK mRNA, is much longer in the mouse '

Figure 1. Genomic context of the human HRK gene. HRK is located on 12 between the RNFT2 and FBXW8 genes. Arrows indicate the 5' to 3' orientation of each gene. Adapted from the NCBI Map Viewer.

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Figure 2. HRK gene and promoter structure. A. Structure of the HRK gene. The structure of the human and rat HRK genes is shown. The HRK gene consists of two exons separated by a large intron. The transcriptional start site is indicated as +1 (see panel B for DNA sequence). Exon 1 contains the HRK open reading frame (black box) as well as a small region of the 3' UTR, the remainder of which is in exon 2. Exon 2 is longer in the rat and mouse genes compared to Exon 2 in human HRK. The human HRK transcript is 716 nucleotides long (Inohara et al., 1997) whereas the rat HRK transcript is 5253 nucleotides long due to the larger size of Exon 2 (Imaizumi et al., 1997). B. HRK promoter sequence. Alignment of the promoter sequences for the rat, mouse, human and cow HRK genes. Shaded regions indicate a conserved ATF site, a GC box, an E box and a TATA box. * represent bases conserved in all four species. Overall, 80% of the nucleotides are conserved. The transcriptional start site of the rat HRK gene determined by 5' RACE is indicated as +1, together with the direction of transcription (Towers et al., 2009). The HRK promoter has a similar structure in the four species except that there is a 10 bp deletion between the ATF site and putative GC box in the human gene, compared to the other species. Adapted from Figure 3 in Towers et al. (2009). and rat genes compared to human HRK (see Figure liver, lung, testis, heart, intestine or skeletal muscle 2A). In the three species only one major transcript (Imaizumi et al., 1997; Coultas et al., 2007). encoding a single protein isoform has been described. Based on studies with the rat HRK gene (Ma et al., Transcription 2007; Towers et al., 2009), the 1 kb region upstream of exon 1 contains elements important for the control of In northern blotting experiments with RNA from HRK transcription (Figure 2B). The HRK promoter is human tissues, it was reported that the HRK transcript GC-rich but contains a conserved block of 14 A/T was detected in spleen, lymph nodes, thymus, bone nucleotides that might function as a TATA box, a marrow and appendix (Inohara et al., 1997). However, conserved E-box, a conserved GC box and a conserved in the rat and mouse the expression of the HRK and functionally-important ATF binding site (5'- transcript is much more restricted and HRK is detected TGATGTAA-3') that binds c-Jun and ATF2 and which in the brain but not the spleen, thymus, bone marrow, is important for the

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Figure 3. Structure of the human and rat HRK proteins. Amino acid residues in the 91 amino acid human HRK protein are numbered. Residues that are identical in the human and rat HRK proteins are shaded. Gaps are indicated by -. The BH3 domain and transmembrane domain are marked by black lines. activation of HRK transcription by the JNK pathway HRK predominantly localizes to mitochondria following survival factor withdrawal in neurons (Ma et (Sunayama et al., 2004). Studies with a 27 amino acid al., 2007; Towers et al., 2009) or exposure to pro- peptide containing the putative transmembrane domain inflammatory cytokines in pancreatic beta-cells of HRK indicated that this domain is able (Gurzov et al., 2009). to insert into membranes, where it adopts a Pseudogene transmembrane alpha-helical structure (Bernabeu et al., 2007). This suggests that the carboxy terminal region There are no known pseudogenes for HRK. of HRK may insert into the mitochondrial outer Protein membrane. Function Description HRK is a pro-apoptotic BH3-only member of the Bcl-2 Only one isoform of the HRK protein has been protein family. Overexpression of HRK can induce described. HRK is 91 amino acids long in humans and apoptosis in HEK293 cells (Inohara et al., 1997; 92 amino acids in mouse and rat (Inohara et al., 1997; Imaizumi et al., 1999), rat sympathetic neurons Imaizumi et al., 1997; Imaizumi et al., 1999). (Imaizumi et al., 1997) and cerebellar granule neurons (Harris and Johnson, 2001). In these cell types Expression HRK/DP5-induced apoptosis is blocked by co- See section on transcription for information about expression of Bcl-2 or Bcl-xL (Inohara et al., 1997; tissue specificity. The endogenous HRK protein has Imaizumi et al., 1997) or by knockout of the been detected in immunoblotting experiments with a endogenous Bax gene (Harris and Johnson, 2001). The number of cell types, for example: in NGF-deprived rat HRK protein contains two functional domains: a BH3 primary sympathetic neurons in culture (Imaizumi et domain and a carboxy terminal transmembrane domain al., 1997), in the mouse brain following focal cerebral (Figure 3). The HRK BH3 domain is related in amino ischemia (middle cerebral artery occlusion; Gao et al., acid sequence to the BH3 domains of other Bcl-2 2005), in the auditory cell line HEI-OC1 exposed to family proteins and is required for interaction with anti- gentimicin (Kalinec et al., 2005) and in the pancreatic apoptotic Bcl-2 family proteins, such as Bcl-2 and Bcl- beta-cell line INS-1E treated with the pro-inflammatory xL, and for cell death induced by overexpression of cytokines IL-1beta and IFNgamma (Gurzov et al., HRK (Inohara et al., 1997). A detailed study of the 2009). binding of BH3-only proteins to anti-apoptotic Bcl-2 Localisation family members indicated that HRK binds with high affinity to Bcl-xL, Bcl-w and A1 and with moderate HRK is a non-nuclear intracellular protein (Inohara et affinity to Bcl-2 and Mcl-1 (Chen et al., 2005). The al., 1997). Flag-tagged HRK co-localizes with HRK transmembrane domain is rich in hydrophobic MitoTracker in transfected COS-7 cells suggesting that amino acid residues and could mediate the insertion of

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the HRK carboxy terminus into intracellular secondary glioblastomas as compared to other types of membranes, such as the mitrochondrial outer tumours, and aberrant methylation was closely membrane. associated with loss of expression. Reverse Homology transcription-PCR analysis also demonstrated a clear agreement between reduced HRK protein levels and The HRK protein is only related to other Bcl-2 family low or absent HRK transcripts. proteins in the short BH3 domain. Disease Mutations Glioblastoma (WHO grade IV) is the most frequent and most malignant tumour of the human nervous system. Note Despite advances in surgery and adjuvant therapy, No mutations have been described in HRK. glioblastoma patients still have a very poor prognosis. From a clinical and biological point of view, Implicated in glioblastomas are divided into two subtypes. Primary or de novo glioblastomas develop rapidly, without clinical Colorectal and gastric cancer or histopathological evidence of less malignant Note precursor lesions and constitute the majority of The region around the HRK transcriptional start site diagnosed cases, whereas secondary glioblastoma was methylated in 36% of colorectal and 32% of develops more slowly and progressively from low- gastric cancer cell lines and was closely associated with grade diffuse (WHO grade II) or anaplastic (WHO a loss of HRK expression in those cell lines (Obata et grade III) astrocytoma. al., 2003; Nakamura et al., 2009). HRK expression was restored by treatment with a methyltransferase References inhibitor, 5-aza-deoxycytidine, and further enhanced by Imaizumi K, Tsuda M, Imai Y, Wanaka A, Takagi T, Tohyama addition of the histone deacetylase inhibitor trichostatin M. Molecular cloning of a novel polypeptide, DP5, induced A or depsipeptide. The restoration of HRK expression during programmed neuronal death. J Biol Chem. 1997 Jul correlated with an induction of apoptosis and 25;272(30):18842-8 enhancement of Adriamycin-induced apoptosis. Inohara N, Ding L, Chen S, Núñez G. harakiri, a novel Expression of other proapoptotic genes, including regulator of cell death, encodes a protein that activates apoptosis and interacts selectively with survival-promoting BAX, BAD, BID, and PUMA, was unaffected by proteins Bcl-2 and Bcl-X(L). EMBO J. 1997 Apr 1;16(7):1686- treatment with 5-aza-deoxycytidine. Aberrant 94 methylation of HRK was also frequently detected in Imaizumi K, Morihara T, Mori Y, Katayama T, Tsuda M, primary colorectal cancers that showed methylation of Furuyama T, Wanaka A, Takeda M, Tohyama M. The cell multiple genes. death-promoting gene DP5, which interacts with the BCL2 family, is induced during neuronal apoptosis following Disease exposure to amyloid beta protein. J Biol Chem. 1999 Mar Colorectal cancer (bowel cancer) is characterized by 19;274(12):7975-81 neoplasia in the colon, rectum or vermiform appendix. Harris CA, Johnson EM Jr. BH3-only Bcl-2 family members are Colorectal cancers start in the lining of the bowel. If coordinately regulated by the JNK pathway and require Bax to left untreated, they can grow into the muscle layers induce apoptosis in neurons. J Biol Chem. 2001 Oct underneath, and then through the bowel wall. Most 12;276(41):37754-60 begin as a small growth on the bowel wall - a colorectal Obata T, Toyota M, Satoh A, Sasaki Y, Ogi K, Akino K, Suzuki polyp or adenoma. These growths are usually benign, H, Murai M, Kikuchi T, Mita H, Itoh F, Issa JP, Tokino T, Imai but some develop into cancer over time. Colorectal K. Identification of HRK as a target of epigenetic inactivation in colorectal and gastric cancer. Clin Cancer Res. 2003 Dec cancer is the third most commonly diagnosed cancer in 15;9(17):6410-8 the world. Gastric cancer (stomach cancer) can develop in any Imaizumi K, Benito A, Kiryu-Seo S, Gonzalez V, Inohara N, Lieberman AP, Kiyama H, Nuñez G. Critical role for part of the stomach and may spread throughout the DP5/Harakiri, a Bcl-2 homology domain 3-only Bcl-2 family stomach and to other organs, in particular the member, in axotomy-induced neuronal cell death. J Neurosci. oesophagus, lungs, lymph nodes and the liver. Stomach 2004 Apr 14;24(15):3721-5 cancer causes about 800000 deaths worldwide per year. Sunayama J, Ando Y, Itoh N, Tomiyama A, Sakurada K, Sugiyama A, Kang D, Tashiro F, Gotoh Y, Kuchino Y, Kitanaka Glioblastoma C. Physical and functional interaction between BH3-only Note protein Hrk and mitochondrial pore-forming protein p32. Cell The region around the HRK transcriptional start site Death Differ. 2004 Jul;11(7):771-81 was methylated in 19% of diffuse astrocytomas, in 22% Chen L, Willis SN, Wei A, Smith BJ, Fletcher JI, Hinds MG, of anaplastic astrocytomas, in 27% of primary Colman PM, Day CL, Adams JM, Huang DC. Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands glioblastomas, and in 43% of secondary glioblastomas allows complementary apoptotic function. Mol Cell. 2005 Feb (Nakamaura et al., 2005; Nakamura et al., 2009). HRK 4;17(3):393-403 expression was significantly reduced in 61% of

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Gao Y, Signore AP, Yin W, Cao G, Yin XM, Sun F, Luo Y, required for apoptosis induced by potassium deprivation in Graham SH, Chen J. Neuroprotection against focal ischemic cerebellar granule neurons. J Biol Chem. 2007 Oct brain injury by inhibition of c-Jun N-terminal kinase and 19;282(42):30901-9 attenuation of the mitochondrial apoptosis-signaling pathway. J Cereb Blood Flow Metab. 2005 Jun;25(6):694-712 Nakamura M, Shimada K, Konishi N. The role of HRK gene in human cancer. Oncogene. 2008 Dec;27 Suppl 1:S105-13 Kalinec GM, Fernandez-Zapico ME, Urrutia R, Esteban- Cruciani N, Chen S, Kalinec F. Pivotal role of Harakiri in the Gurzov EN, Ortis F, Cunha DA, Gosset G, Li M, Cardozo AK, induction and prevention of gentamicin-induced hearing loss. Eizirik DL. Signaling by IL-1beta+IFN-gamma and ER stress Proc Natl Acad Sci U S A. 2005 Nov 1;102(44):16019-24 converge on DP5/Hrk activation: a novel mechanism for pancreatic beta-cell apoptosis. Cell Death Differ. 2009 Nakamura M, Ishida E, Shimada K, Nakase H, Sakaki T, Nov;16(11):1539-50 Konishi N. Frequent HRK inactivation associated with low apoptotic index in secondary glioblastomas. Acta Neuropathol. Towers E, Gilley J, Randall R, Hughes R, Kristiansen M, Ham 2005 Oct;110(4):402-10 J. The proapoptotic dp5 gene is a direct target of the MLK- JNK-c-Jun pathway in sympathetic neurons. Nucleic Acids Bernabeu A, Guillén J, Pérez-Berná AJ, Moreno MR, Villalaín Res. 2009 May;37(9):3044-60 J. Structure of the C-terminal domain of the pro-apoptotic protein Hrk and its interaction with model membranes. Biochim Chipuk JE, Moldoveanu T, Llambi F, Parsons MJ, Green DR. Biophys Acta. 2007 Jun;1768(6):1659-70 The BCL-2 family reunion. Mol Cell. 2010 Feb 12;37(3):299- 310 Coultas L, Terzano S, Thomas T, Voss A, Reid K, Stanley EG, Scott CL, Bouillet P, Bartlett P, Ham J, Adams JM, Strasser A. This article should be referenced as such: Hrk/DP5 contributes to the apoptosis of select neuronal populations but is dispensable for haematopoietic cell Ham J. HRK (harakiri, BCL2 interacting protein (contains only apoptosis. J Cell Sci. 2007 Jun 15;120(Pt 12):2044-52 BH3 domain)). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(12):1033-1037. Ma C, Ying C, Yuan Z, Song B, Li D, Liu Y, Lai B, Li W, Chen R, Ching YP, Li M. dp5/HRK is a c-Jun target gene and

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