Determination of human tissue expression of alternate DDAH-1 transcript

transcript and the alternate T2 transcript in a human tissue cDNA panel (Clontech Laboratories, USA). This cDNA panel from a variety of human tissues is normalized to several different house-keeping genes to ensure accurate relative assessment of

target mRNA abundance.

Taqman qPCR probes (Life Tech Inc, USA) were used to distinguish the two transcripts – an initial probe detecting a 77bp amplicon across the exon 2-3 boundary was used to detect both the T1 and T2 transcripts, and a second probe detecting a 71bp amplicon across the exon 1-2 boundary of the T2 transcript was also used – this was selective for the T2 transcript (Figure 5.3).

As shown in figure 5.4 although a broad distribution of expression for the full length T1 DDAH-1 transcript was seen, no expression of the T2 transcript was seen in the tissues represented in the cDNA panel. Additionally, no expression of the T2 transcript was seen in human liver samples from patients with cirrhosis (the same samples as examined in chapter 4 section – data not shown).

Figure 5.3: Schematic diagram of promoters and exons of DDAH-1 T1 and T2 transcripts. The T1 transcript is a shorter mRNA, but with 6 coding exons encoding a longer protein. The T2 transcript is a longer mRNA but has only 5 coding exons, with 2 non-coding exons, and is therefore predicted to encode a shorter protein.

Since, endometrial tissue was not represented in this cDNA panel, and the original curated NCBI entry for this transcript was from an endometrial cancer cell line, RNA from the Ishikawa endometrial cell line was also analysed by qPCR for the T2

transcript. As seen in figure 5.4 low level expression of the T2 transcript is evident. Although a direct comparison with the mRNA expression of the T1 transcript is not possible since the ΔΔCT method was used for RNA quantification, rather than standard curve method, it is clearly lower than the full-length T1 transcript. No expression of T2 was seen in the negative controls for this experiment.

Figure 5.4: Left panel: DDAH-1 T1 transcript mRNA expression in human tissue cDNA panel (pooled cDNA from n=6 tissues/group) . No DDAH-1 T2 transcript expression was detectable in this tissue panel. Right panel: Expression of both DDAH-1 T1 and T2 transcript mRNA is detectable in the Ishikawa cell line (pooled RNA from n=4 experiments).

To confirm these findings, healthy human placental tissue (n=4) was obtained from healthy mothers at the time of caesarean section. Non-targeted samples were taken, snap frozen in liquid nitrogen, and RNA extracted for qPCR as in section 2.4.1. As shown in figure 5.5 there is similarly low-level expression of the T2 mRNA transcript seen in healthy placenta, although again direct comparison with the expression level of the T1 transcript is not possible with the ΔΔCT method of quantification. Again, no expression of T2 was seen in the negative controls for this experiment.

DDAH-1 T1 transcript DDAH-1 T2 transcript 0 5 10 15 20 25 2 ^- D D C T (H sD D A H 1 P P IA )

Figure 5.5: DDAH-1 T1 and T2 transcript mRNA expression in healthy human placenta (n=4).

The core promoter is required for the transcription of eukaryotic RNA polymerase II (RNA pol II) transcribed genes. The promoter is typically defined as the area of genomic DNA approximately 35-40 bp upstream and downstream of the TSS. The core promoter is also defined in functional terms, since this region is usually sufficient to mediate gene expression in a reporter gene assay. The core promoter contains sequence elements, referred to as “core promoter motifs,” which interact with the basal transcription machinery, including RNA polymerase II and the TFIID complex199, 200. Although a number of prevalent core promoter motifs have been defined, there is no universal motif common to all promoters. However, the traditional model of a eukaryotic promoter driven by RNA pol II is an AT-rich DNA sequence (the TATA box) approximately 30 bp upstream of an initiator (Inr) sequence that contains the TSS. Assembly of a pre-initiation complex (PIC), which includes the transcription factor TFIIA-H along with RNA pol II, is initiated by TFIID binding to the TATA box, Inr sequences and/or other sites, and bending the DNA through a 90° angle. The next step involves recruitment other general transcription factors, after

which transcription is initiated 30 bp downstream.

Transcription is further regulated by interactions of the PIC with three additional components: the TATA-associated factors, the so-called mediator complex(es), and positive and negative cofactors. Coordination of chromatin modification, mainly through the control of post-translational modification of histones, also has an important role in transcription initiation. The recruitment of all of these co-activators and co-repressors of transcription initiation is controlled by transcription factor binding to cis-acting DNA sequences that can lie within the core promoter or in more remote locations (enhancers and repressors).

Using the Genomatix Gene2Promoter and Matinspector software, a number of transcription factor binding sites were mapped to this putative promoter using default parameters for core and matrix similarity thresholds (matrix similarity values of 0.75 and core similarity values of 0.7)201, 202.

The graphical output identifies 251 significantly conserved TFBS according the above parameters, within the conserved ≈700bp area upstream of the T2 transcript TSS. Specifically, binding sites for important initiators of transcription such as the TATA box (O$VTBP) and TFIID (O$TF2D) are present in this conserved area (Figure 5.6).

Figure 5.6 Identification of putative promoter elements within conserved non-coding element using Genomatix Gene2Promoter. TATA= O$VTBP, TFIID =O$TF2D.

As mentioned above, the TATA box is not a ubiquitous feature of core promoters, but tends to be prevalent in ‘sharp’ or ‘narrow’ promoters. Large-scale mapping studies have revealed that the TSS is usually not a single distinct genomic position; instead, a typical core promoter may consist of an array of closely located TSSs spread over 50–100 bp. Thus, ‘sharp’ or ‘narrow’ peak promoters are distinguished by a tight cluster of TSSs spanning only one or several bps, whereas ‘broad’, or ‘wide’ peak promoters the TSSs are distributed over a wide range, up to 100 bp203_{. Sharp} promoters are typically involved in tissue-specific regulation of expression rather than ‘broad’ promoters which are common in constitutively expressed genes. Therefore, this TATA box located 34bp upstream of the TSS for the T2 DDAH-1 transcript, in a highly evolutionarily conserved non-coding region, supports the hypothesis that this region represents a promoter involved in the tissue-specific regulation of this transcript.

LOCUS GXP_1817791(dimethylarginine dimethylaminohydrolase 1/human) 818 bp

DNA DEFINITION loc=GXL_77723|sym=dimethylarginine dimethylaminohydrolase 1|

geneid=23576|acc=GXP_1817791|taxid=9606|spec=Homo sapiens|

len=818|descr=DDAH1|comm=CompGen promoter ACCESSION GXP_1817791 BASE COUNT 207 a 173 c 220 g 218 t ORIGIN

1 GACGGGGGGC GGGAGGGGGG AGGAGGGGTG CAGGGACAGT ACTGGTTGAA CTAGAGAGAG 61 AATGGAGAGG AGGGTTCCTG GCTTCTGTTG TGGCGTCTTT TCTATTTGAC TTCATGGTTG 121 TAAGTATTTC CAGATGGTGA ATCAGACACC AGACGTAACA ATATTTCCAT ATTTGGGTAT 181 AGCAGTAGCC TGCGTTTTTA ACATTTTTCT GCCTCTGTTA TCCAACCACA AAGCATTCTT 241 GACAGCTTCA AATGTTGTTA AATATAGATT TAACTTCTCT TCCCAGAGCA GGAAATTCTT 301 TGGAATTCCT TGTTTTTCAC GCAATCTGTC CATCATGATT TAAAATAAAA GCACAGTGGA 361 TCATCCAACT GGCCGTATAT ACCTTAATTG GAGGTTGGGG ATGGGGGACG GCAGAGATCC 421 AGTCTGCCGC ACTGCGTTCA AACACACGCC ATTCCAGAGA TTCCTTTAAA ATCACATTAA 481 AGTTTTTTTA ACAAGGGTGT GTGGGTTTGT TTCTGGACTT CAACTGGGGA ATCTTGAGGA 541 TGAGTTTGCC CCAGAAGAGA AATTTAGAGA ACCTTACCGT CAGCTGCCCA TTTAAAGCAG 601 GGGGTGTGTT GTGGGATGGG GGTGGGAAGC TGGAGCAACA GGGCCAGGAG GTGTGGGAGC 661 GGGAGACACT AGAGTAACCT ATGTGCACAG CCTCTCCATA TACCATGTGC TGTTGCGCCT 721 GCTAGTAATC GACGACATTA GGCAAGAGAA ACAGCGGCTC CTCAAGTCCT GCCCAAAGAC 781 CGTCCAGAAA CCCCAGCCTC CCGTCGCCTT CTCGCCGC

Figure 5.7: Sequence of putative promoter of DDAH-1 T2 transcript. Purple=TATA box. Green= coding sequence.

To further explore the hypothesis of endometrial/placental restricted expression of this T2 transcript, the putative promoter was interrogated for transcription factor binding sites that are upregulated in placental tissue in pre-eclampsia. Since pre- eclampsia is a condition characterized by placental vascular dysfunction, elevated placental ADMA and decreased placental DDAH activity, it was hypothesized that pre-eclampsia may be associated with a switch in transcription of full-length DDAH-1 to truncated, non-functional T2 DDAH-1 through the putative alternative promoter of T2.

The transcription factor profile of pre-eclampsia has been previously defined - Vaiman et al recently analysed data from publicly available microarray analyses of pre-eclamptic and normal placentae, to obtain a consensus list of modified genes204_. Subsequently, they demonstrated 67 up-regulated and 31 down-regulated genes, and went on to find over-represented transcription factor binding sites in the promoters of these genes. This group found that the promoters of up-regulated genes are enriched in putative binding sites for NFkB, cAMP responsive element

binding protein 1 (CREB), RAS-responsive element binding protein 1 (RREB1), and

activator protein-2 (AP-2).

When these specific transcription factor sites were searched for in the putative DDAH-1 T2 promoter, several predicted binding sites for CREB and RREB1 are found within the region +/100bp of the TSS, in the vicinity of the proposed TFIID binding site (figure 5.8).

Figure 5.8 Identification of putative transcription factor binding sites within the conserved predicted promoter site of the DDAH-1 T2 transcript using Genomatix Gene2Promoter (accessed May 2016). TFIID=O$TF2D, TATA=$VTBP, CREB=V$CREB, RREB1=V$RREB.

Figure 5.9 Identification of putative transcription factor binding sites within the promoter site of the DDAH-1 T1 transcript using Genomatix Gene2Promoter (accessed May 2016). CREB=V$CREB, AP-2=V$AP2F.

By contrast, in the DDAH-1 T1 transcript promoter, from the above list of