C2H2‐171 : A novel human cDNA representing a developmentally regulated poz domain/zinc finger protein preferentially expressed in brain

We describe a novel human zinc finger cNDA, C2H2‐171. This cDNA represents an mRNA which encodes a protein of 484 amino acids and a calculated molecular weight of 54 kD. Four zinc finger‐like domains are found in the C‐terminal end of the protein. At the N‐terminus, C2H2‐171 contains a POZ/tramtrack‐like domain similar to that found in the tumor associated zinc finger proteins LAZ‐3/BCL‐6 and PLZ‐F, as well as in non‐zinc finger proteins. C2H2‐171 RNA is preferentially expressed in the brain, and increases during the course of murine development, with maximal expression in the adult. C2H2‐171 RNA is differentially expressed in brain regions, with the highest level of expression in the cerebellum. C2H2‐171 RNA was expressed at high levels in primary cerebellar granule cell neurons compared to astrocytes. The gene encoding C2H2‐171 is highly conserved in vertebrates, and maps to the terminus of human chromosome 1 (1q44‐ter). This chromosomal location is associated with a number of cytogenetic aberrations including those involving brain developmental anomalies and tumorigenesis. These data suggest that C2H2‐171 may play an important role in vertebrate brain development and function.

Genes encoding C1H1!type zinc _nger containing proteins comprise one of the largest families in the human genome[ 3\4\7 First described in Xenopus TFIIIA\ 8\18 C1H1 type zinc _nger proteins have been shown to act on the molecular level as transcriptional activators\ 12 or repressors\ 27 and to be involved in chromatin assembly [ 06 In addition\ zinc!_nger proteins have been demonstrated to function in developmental processes 06 and tumorigenesis [ 08 In addition to DNA binding domains\ some zinc _nger genes contain other conserved motifs\ including FAX\ 15 KRAB\ 4 tramtrack\ 11 and homeodomains[ 07 The POZ:tramtrack domain is an amino!terminal conserved domain found in a number of cellular and viral proteins[ 2 In Drosophila\ this domain has been shown to be involved in development\ metamorphosis\ and pattern formation[ 03\11 On the molecular level\ the POZ domain may act as a mediator of proteinÐprotein interaction\ and in some cases\ may function as an inhibitor of DNA binding[ 2 The POZ domain encoding regions of two POZ:zinc _nger containing genes has also been associated with chro! mosomal translocations in non!Hodgkins lymphoma "LAZ!2:BCL5# 14\17\34 and in acute pro! myelocytic leukemia [ 00 In this study\ we describe a novel human cDNA\ C1H1!060\ which represents an mRNA that encodes a protein containing both zinc _nger and POZ!like domains[ The gene encoding C1H1!060 is conserved in vertebrates\ and maps to the terminus of human chromosome 0 "0q33!ter#\ a genetic region associated with developmental anomalies and human tumors[ C1H1!060 RNA is preferentially expressed in the brain\ and within the brain is expressed di}erentially\ with highest levels of expression in the cerebellum[ The RNA is expressed at higher levels in primary cultures of neurons than astrocytes[ In addition\ C1H1!060 RNA expression increases during development[

Isolation of cDNA clones
A human hippocampal cDNA library from a normal 1!year!old female in lambda!ZAP II vector "Stratagene è825194# was screened with a degenerate oligonucleotide speci_c for a conserved amino acid element "TGEKP# found in C1H1!type zinc _nger proteins\ as previously described[ 3 Two overlapping clones\ C1H1!060 and C1H1!36 were obtained for full length cDNA sequencing[

Automatic DNA sequencing
Plasmid was prepared for sequencing using Qiawell 7!Plus "Qiagen\ Inc[# as described by the manufacturer[ Double stranded cDNA clones in pBluescript SK − vector "Stratagene\ Inc[# were sequenced in both directions by creating nested deletions followed by sequence analysis[ Sequencing reactions were performed on an Applied Biosystems robotic workstation and analyzed on an Applied Biosystems 262A automated DNA sequencer using~uorescent labeled vector primers[

Database comparisons
DNA and protein searches were performed using the BLAST 1 network server at the National Center for Biotechnology Information "NCBI# against the DNA and protein non!redundant data! bases[ Local sequences alignments were performed with the GCG based program FASTA[ 02

Southern analysis
Genomic DNA from various species was digested with EcoRI and separated by gel electrophoresis through a 0) agarose gel and transferred to nylon as described[ 23 C1H1!060 cDNA insert was labeled with 21 P by random priming "Prime!It\ Stratagene#[ Southern blots were hybridized for 1 h at 58>C in Quikhyb "Stratagene# and washed for 29 min at room temperature with 1×SSC and 9[0) SDS\ followed by washing at 44>C with 0×SSC and 9[0) SDS[ The _lter was then dried and exposed to X!ray _lm at −79>C[

Chromosomal localization
PCR primers were designed to amplify a 036 bp product from the 2? UTR of C1H1!060 cDNA

Isolation of a novel cDNA representin`an RNA which encodes a protein containin`novel POZ and zinc _n`er!line domains
A human hippocampal cDNA library from a normal 1!year!old female was screened with a degenerate oligonucleotide speci_c for C1H1!type zinc _nger genes[ 3 By partial expressed sequence tag "EST# sequence analysis\ it was determined that 19 independent clones represented fragments of the same novel zinc _nger coding cDNA[ Two independent overlapping cDNA clones\ C1H1!36 and C1H1!060\ were sequenced in both directions[ The sequence reveals a cDNA of 1905 nucleotides\ as presented in Fig[ 0 !060 RNA is selectively expressed in adult brain compared to other tissues examined[ However\ C1H1!060 may exist in non!brain tissues at concentrations below the level of detection of the Northern blot analyses described here[ C1H1!060 appears to play a regionally speci_c function within the brain\ since it is di}erentially expressed in brain regions\ with maximal expression observed in the cerebellum[ C1H1!060 RNA is preferentially expressed in primary neuron cultures relative to astrocytes[ However\ it is possible that it may be expressed in astrocytes at di}erent stages of development and in other glia\ including microglia and oligodendrocytes[ This might explain the low levels of RNA detected in the corpus callosum\ a structure composed primarily of glia and myelinated axons[ Northern analysis also revealed that C1H1!060 RNA is absent in early devel! opment in both head and body tissues[ RNA levels are detectable in the head at approximately day 04 in murine embryonic development\ increase through birth\ and are maximal in adult brain[ This expression pattern may indicate that C1H1!060 functions in late stage brain development and in the adult nervous system[ Interestingly\ neurons of the deep cerebellar nuclei as well as cerebellar Purkinje cell neurons are generated near embryonic day 04 in the rodent[ 0 Likewise\ a variety of morphological changes are occurring in the cerebellum during this stage of development including the di}erentiation of cerebellar granule cell neurons [ 0 The evolutionary conservation of C1H1!060 was analyzed by cross species Southern blot hybrid! ization[ C1H1!060 shows equivalent hybridization signal strength among vertebrates[ This pattern is consistent with the _rst of two Southern blot patterns seen in similar evolutionary studies of C1H1!type zinc _nger genes[ These patterns are ] "a# equivalent hybridization signal strength in vertebrates or\ "b# strong conservation in primates only[ 20 The observation that C1H1!060 gene is not present in Drosophila and yeast is consistent with C1H1!060 being important in vertebrate brain development[ C1H1!060 maps to the distal portion of chromosome 0 "0q33!ter# by PCR analysis of MEGA! YAC clones[ Another C1H1 type zinc _nger gene\ HZF!05\ also maps to this region[ 25 Chromosomal duplication and deletion in this region is associated with numerous congenital anomalies including brain developmental anomalies[ 05 These include mental retardation\ microcephaly\ and autistic!like behavior[ 10\13\29\28 Chromosomal abnormalities in this region have also been associated with a number of human tumors including Wilms tumor\ 31 astrocytomas 31 and AML\ 22 and with congenital ocular defects[ 01 Further analysis of this gene on the distal arm of chromosome 0q may provide clues to a possible role in brain development or tumorigenesis[