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Am J Physiol Renal Physiol 294: F525-F533, 2008. First published January 9, 2008; doi:10.1152/ajprenal.00299.2007
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Transcription of laminin {gamma}1 chain gene in rat mesangial cells: constitutive and inducible RNA polymerase II recruitment and chromatin states

Joel D. Nelson,1 Steve Flanagin,2 Yasunobu Kawata,2 Oleg Denisenko,2 and Karol Bomsztyk1,2

1Molecular and Cellular Biology Program and 2Department of Medicine, University of Washington Medicine Lake Union, Seattle, Washington

Submitted 29 June 2007 ; accepted in final form 2 January 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The laminin {gamma}1 chain, a critical component of the extracellular matrix, is encoded by the 125-kb-long Lamc1 locus. We profiled RNA polymerase II (Pol II) and histone modifications along the Lamc1 locus to explore transcription of this gene in its native chromatin environment. Treatment with 12-O-tetradecanoylphorbol-13-acetate increased Lamc1 mRNA in rat mesangial cells (RMC). This increase was matched by an increase in Pol II density along the entire length of the Lamc1 locus. In contrast, in the hepatocarcinoma cell line (HTC-IR) an increase in Pol II density was restricted to the promoter and was not followed by mRNA induction. The pattern of histone H3 methylation was similar for both cell types but an increase in H3 lysine 9 acetylation observed at the 5'-end was weaker in HTC-IR cells than in RMC. All of the histone modifications showed spatial patterns where levels differed greatly between the 5'- and 3'-ends of Lamc1. Conversely, at the short, highly induced egr-1 gene the differences in chromatin marks between the 5'- and 3'-ends were much smaller. The results of this study suggest that 1) Lamc1 transcription can be controlled after transcription initiation, to our knowledge, the first time this has been shown in an extracellular matrix gene, and 2) the length of a gene is a factor that can affect the chromatin environment for Pol II elongation.

Fast ChIP; extracellular matrix; hnRNP K; histone; elongation; egr-1

GENE TRANSCRIPTION is composed of a sequence of complex events, which have been conceptually viewed as three discreet phases: initiation, elongation, and termination. Transcription initiation is the most studied phase, and it consists of highly regulated steps that result in the assembly of general transcription factors (GTFs) and RNA polymerase II (Pol II) into a preinitiation complex (PIC) at the core promoter (63). Transcription elongation is a process that follows initiation by which Pol II moves through the gene and synthesizes RNA (57). The termination step consists of cleavage of nascent mRNA, polyadenylation of the mRNA 3'-end, and its release from the DNA template.

Laminins are major basement membrane proteins that form heterotrimers composed of {alpha}, β, and {gamma} chains (3). In the extracellular matrix (ECM), the {gamma}1 chain, which is found in 10 of the 16 known trimeric laminin isoforms, is the most widely expressed laminin chain (3, 64). Laminin {gamma}1 is required for endodermal differentiation and its absence causes early lethality in mouse embryos (40, 59). Also, expression of laminin {gamma}1 is increased in cultured mesangial cells exposed to high concentrations of glucose (47, 50) and is one component of the increased ECM expression seen in models of diabetic glomerulosclerosis (15). These and other observations suggest that the molecular composition of laminins is critical (2, 36), that misexpression of laminin {gamma}1 may be a factor in renal disease, and that laminin {gamma}1 is an essential component of the ECM which orchestrates developmental events (40, 59).

Because of their importance, expression of {gamma}1 and other laminin chains (1, 64) has been extensively explored. Most of these studies have focused on promoter elements and factors that initiate their transcription (19, 21, 31, 45) or regulate their posttranscriptional processing (66). Regulation of transcriptional elongation of not only the laminin genes but also other components of the ECM has not been examined. In fact, most studies on transcriptional elongation have been carried out either in vitro (5) or in yeast (32, 58).

It has been estimated that 75–90% of eukaryotic genomic DNA is wrapped in nucleosomes composed of histones (55). Nucleosomes present an obstacle for transcribing Pol II (61). The ability of Pol II to elongate through the nucleosomal array is partly dependent on remodeling factors (39) and histone-modifying enzymes (16, 24, 35, 57) that either travel with RNA Pol II or bind directly to chromatin (68). Pol II density is highest at the start of the gene and progressively decreases toward the 3'-end (4, 18, 25). The different density of Pol II along the genes may, in part, explain the observation that chromatin state differs between the 5'- to 3'-ends of transcribed regions (6, 56). For example, histone H3 lysine 4 trimethylation, a modification that recruits ATP-dependent chromatin remodeling factors, is highest in the 5' regions of transcribed genes (68). These remodeling factors destabilize nucleosomes at the start of a gene, which is thought to facilitate the early phase of elongation.

ECM genes, like Lamc1, are typically much longer than the average mammalian gene (~25 kbp). It is conceivable that as the length of a gene increases and the ends become linearly more isolated from each other, the gradient of Pol II and histone modification differences between the two ends increases. This, in turn, would expose elongating Pol II to larger extremes of chromatin state from one end of the gene to the other such that the rate of elongation could be significantly different at the beginning and the end of a long gene. To explore this issue, we used quantitative chromatin immunoprecipitation assays (41) to follow inducible Pol II recruitment and chromatin states along the 125-kb-long Lamc1 gene and compared them to the short potently inducible immediate early gene egr-1.

It is becoming increasingly apparent that level of transcription of the same gene in different cell types of the same species or the same cell type in different species is achieved by different mechanisms (42). Thus, in addition to examining differences in elongation of different genes (i.e., long vs. short), we also were interested in whether elongation of the same gene could differ between cell types. We used ChIP assays to follow Pol II and chromatin states in two cell types where expression of Lamc1 differs.


   METHODS AND MATERIALS
TOP
 ABSTRACT
 METHODS AND MATERIALS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mammalian Cells

Rat mesangial cells (RMC; ATCC no. CRL-2573) were grown in 150-mm plastic cell culture dishes in RPMI 1640 media supplemented with 10% FBS, 2 mM glutamine, penicillin (100 U/ml), streptomycin (0.01%), and humidified with 5–95% CO2-air gas mixture (62). Forty-eight hours before 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment, the culture medium was switched to the starvation medium: RPMI 1640 with 0.5% FBS, 2 mM glutamine, penicillin (100 U/ml), and streptomycin (0.01%). The cells were treated 2 days later with 10–7 M TPA in starvation medium for 60, 120, or 240 min or were left untreated. Hepatocarcinoma cell line (HTC-IR) cells were grown in 100-mm plastic cell culture dishes in DMEM media supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (0.01%), and humidified with 5–95% CO2-air gas mixture (62). Twenty-four hours before TPA treatment, the culture medium was switched to the starvation medium: DMEM with 0.5% FBS, penicillin (100 U/ml), and streptomycin (0.01%). The cells were treated 1 day later with 10–7 M TPA in starvation medium for 60, 120, or 240 min or were left untreated.

Reagents

Antibodies. The antibody to the COOH-terminal peptide of K protein was raised in rabbits as described before (65). Rabbit IgG fraction was from Vector Laboratories (cat. no. I-1000), other antibodies were to RNA Pol II (anti-Pol II, 2 µg per IP, Santa Cruz Biotechnology, cat. no. sc-899), acetyl histone H3 Lys9 (anti-H3-K9Ac, 2 µl per IP, Cell Signaling, cat. no. 9671S), histone H3 (anti-H3, 1 µg per IP, Abcam, cat. no. ab1791), dimethyl H3 Lys4 (anti-H3-K4m2, 1 µl per IP, Upstate Biotechnology, cat. no. 07-030), trimethyl H3 Lys4 (anti-H3-K4m3, 1 µg per IP, Abcam, cat. no. ab8580 or Novus Biologicals, cat. no. NB500-173), trimethyl H3 Lys27 (anti-H3-K27m3, 1 µg per IP, Abcam, cat. no. Ab6002), trimethyl H3 Lys36 (anti-H3-K36m3, 1 µg per IP, Abcam, cat. no. 9050), dimethyl H3 Lys79 (anti-H3-K79m2, 1 µg per IP, Abcam, cat. no. 3594), Chelex-100 was purchased from Bio-Rad (cat. no. 142-1253), and proteinase K from Invitrogen (cat. no. 25530-015).

RNA Isolation and Quantitative RT-PCR

Total cellular RNA was isolated using TRIzol reagent as per manufacturer's protocol (Invitrogen). Reverse transcriptase (RT) reactions were carried out using MMLV RT (Invitrogen) and random hexamer primers with 40 units of RNAse inhibitor (Invitrogen) in a 20-µl volume as per manufacturer's protocol. RT reactions were diluted 1:100 with water before use in PCR reactions. Differences in efficiency between the primer pairs used in real-time PCR were normalized using standard curves. Briefly, real-time PCR reactions were run on diluted cDNAs and four dilutions of RMC genomic DNA (50, 12.5, 3.13, and 0.781 ng total DNA per reaction) for each primer pair. A curve of Ct vs. log DNA concentration was generated for each primer pair (R2 ≥ 0.993) and slope (m) and y-intercept (b) were obtained. Cts from the PCR reactions on the cDNAs were used in the following equation to calculate the relative amounts of the mRNAs: amount = 10Ct·m–b.

Chromatin Immunoprecipitation

The ChIP assay was carried out as previously described (41). RMC from four 15-cm Petri dishes per one time point were used to prepare chromatin for up to 10 precipitations.

Real-Time PCR

The reaction mixture contained 5 µl 2x SYBR Green PCR Master Mix (Applied Biosystems), 2.5 µl DNA template, and 0.3 µM primers (10 µl final volume) in a 384-well Optical Reaction Plate (Applied Biosystems). The 7900HT Real-Time PCR system (3-step protocol, 40 cycles) was used for amplification. Sequences of primers used in these studies are available upon request. Examples of real-time PCR amplification and dissociation curves are illustrated in Fig. S4. (The online version of this article contains supplemental data.)

Data Acquisition, Analysis, and Graphics

PCR data were acquired using SDS Enterprise Database software (Applied Biosystems). Microsoft Excel-based script programs were created to reduce data processing time and to provide consistent and reliable graphical presentation of the data.


   RESULTS
TOP
 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Laminin {gamma}1 mRNA Expression

In RMC, laminin {gamma}1, Lamc1, mRNA is constitutively expressed and the levels increase with TPA treatment (62). In these cells, TPA is also a potent activator of the immediate early response gene, egr-1 (46). The use of TPA provides a way to explore Pol II dynamics and chromatin changes associated with the expression of Lamc1, and to compare it with the short egr-1 gene (Fig. 1).


Figure 1
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  		Fig. 1. Constitutive and inducible Lamc1 and egr-1 mRNA levels in rat mesangial cells (RMC) and hepatocarcinoma cell line (HTC-IR) cells. A: diagrams showing structure of rat Lamc1 (NC_005112.2, 127,966) and egr-1 (NC_005117.2, 3785) genes; exon (boxes), intron (zig-zag lines), and intergenic (straight lines) regions are shown; genes are drawn to the same scale in kb units (ruler). The arrow heads show the location of primer pairs used in RT-PCR. B: serum-deprived RMC were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA; 10–7 M) for the indicated time points. Total cell RNA was used in RT reactions with random hexamer primers. Real-time PCR, done in triplicate, was carried out with primers to the end of either the Lamc1 (exon 28) or egr-1 (exon 2) genes, as well as to β-actin. Relative mRNA levels were calculated as a ratio of the amount of Lamc1 or egr-1 mRNA to β-actin mRNA. The data represent means ± SE of 3 independent experiments. *P < 0.05 vs. untreated (t-test).

 
Figure 1B shows the results of the transcript level analysis by RT-PCR. In quiescent RMC, the levels of Lamc1 mRNA were higher than for egr-1 mRNA. Approximately 2 h after TPA addition to RMC there was a slow induction of Lamc1 expression with a significant, nearly 2.5-fold increase in the mRNA level at 4 h of treatment (Fig. 1B, top left). In contrast, there was a 13-fold transient increase in the egr-1 mRNA level peaking at 1 h after TPA addition (Fig. 1B, bottom left). No mRNA for the control silenced β-globin gene was detected (data not shown). Interestingly, in quiescent HTC-IR cells, Lamc1 expression was comparable to that in RMC; however, it was not affected by TPA treatment (Fig. 1B, top right). This was not due to the cells being insensitive to TPA since egr-1 expression was induced to the same degree as in RMC (Fig. 1B, bottom right). These differences in Lamc1 expression between the two cell lines may reflect differences in either Pol II initiation or elongation, possibilities that were examined next.

Kinetics of TPA-Inducible Pol II Recruitment Along the Lamc1 Locus in RMC and HTC-IR Cells

TPA treatment increased Lamc1 mRNA levels in RMC but not in HTC-IR cells. To examine the differences between these cell lines, we profiled changes in Pol II density along the Lamc1 locus by using ChIP assays. Figure 2 illustrates the kinetics of Pol II recruitment along Lamc1 in both cell types. In RMC, the highest level of Pol II density was a few hundred basepairs downstream of the transcription start site at exon 1, with overall levels decreasing gradually downstream from this site (Fig. 2A). In response to TPA treatment, an approximately twofold increase in Pol II level could be seen at all of the Lamc1 sites, except for the –20-kb flanking site; however, the kinetics of this increase differed from the 5'- to 3'-end. For instance, the highest level of Pol II presence at exon 1 occurred 1 h after TPA treatment, whereas at exon 28, Pol II peaked an hour later. This delay may reflect how long it takes for Pol II to reach the end of this long gene (Fig. 2A, top row). At the promoter in HTC-IR cells, Pol II levels increased nearly twofold after 1 h of TPA treatment; however, this increase was not seen at other sites downstream from the promoter of the gene. The second row in Fig. 2, A and B, shows the kinetics of binding of heterogeneous nuclear ribonucleoprotein K (hnRNP K), which is an RNA binding protein involved in multiple aspects of gene expression (8). After TPA treatment of RMC, hnRNP K recruitment increased with kinetics which were, within the transcribed region of the gene, similar to those of Pol II (Fig. 2A). This was also the case for egr-1 in RMC (Fig. 3). At Lamc1 in HTC-IR cells, however, hnRNP K recruitment was unaffected by TPA (Fig. 2B). It is likely that hnRNP K is involved in multiple steps, such as regulation of Pol II recruitment, elongation, and RNA processing. In the latter case, this may include binding to the nascent transcript. These results suggest that a TPA-inducible increase in Lamc1 mRNA, seen in RMC but not HTC-IR cells, is associated with the ability of inducibly recruited Pol II at the Lamc1 promoter to productively transcribe the gene in RMC, but not HTC-IR cells. This indicates that transcription of the Lamc1 gene can be controlled after transcription initiation, which is in agreement with observations for several other genes (60).


Figure 2
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  		Fig. 2. Inducible recruitment of RNA polymerase II (Pol II), hnRNP K, and changes in histone H3 modifications along the 125-kb Lamc1 gene in TPA-treated RMC and HTC-IR cells. Resting serum-deprived RMC (A) or HTC-IR cells (B) were treated with 10–7 M TPA for the indicated time points and then used in ChIP assays with antibodies to Pol II, hnRNP K, H3K9Ac, and total H3. Purified DNA was used in real-time PCR with pairs of primers spanning the Lamc1 locus. Results are expressed as ratios of the real-time PCR signals obtained for IP with specific antibodies to mock IP (nonimmune IgG). Location of PCR primers relative to transcription start sites (in bp): 5' flanking; –20,034, promoter; –305, Exon (Ex) 1; +454, Intron 1–2; +820, Exon 2; +81,378, and Exon 28 (last exon); +125,232 (positions illustrated in the cartoon above). Insets in the Pol II graphs at exon 2 and exon 28 show the same data with the scale of the y-axis decreased 5-fold. Data represent means from 3 independent experiments for RMC and 2 independent experiments for HTC-IR cells. *P < 0.05 vs. untreated (t-test).

 

Figure 3
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  		Fig. 3. Inducible recruitment of Pol II and hnRNP K protein and chromatin changes along the egr-1 locus in RMC and HTC-IR cells. Resting serum-deprived RMC or HTC-IR cells were treated with 10–7 M TPA for the given time points and then used in ChIP assays with the antibodies to RNA Pol II, hnRNP K, and histone. Purified DNA was used in real-time PCR with pairs of primers spanning the egr-1 locus. Results are expressed as ratios of the real-time PCR signals obtained for IP with specific antibodies to mock IP (nonimmune IgG). Location of PCR primers relative to transcription start sites (in bp): 5' flanking (–5 kb); –5,278, promoter; –200, Exon 1; +310, Exon 2 (last exon); +3,386, 3' flanking (+8 kb); +8772 (positions illustrated in the cartoon above). Data represent means ± SE from 3 independent experiments in RMC and 2 in HTC-IR cells.

 
Histone H3 Modification Profiles and Changes Associated with TPA Induction of Lamc1 in RMC and HTC-IR Cells

The NH2-terminal tails of the histone proteins H2A, H2B, H3, and H4 are substrates for a large number of covalent modifications and specific combinations of some of these marks reflect the transcriptional states of genes (26). We looked at the profiles of a number of well-studied modifications of histone H3 to see whether they could account for the difference in transcription of Lamc1 in the two cell types (Figs. 2, A and B, and S1A and S1B).

Histone H3. Total histone H3 levels along the length of Lamc1 were affected little by TPA treatment in both cells types (Fig. 2, A and B). The lowest overall levels were at the promoter which is consistent with a previously described ~600-bp stretch of DNase I hypersensitivity in this region indicating nucleosome-free DNA (12). In addition, lower nucleosome density in regions flanking transcription start sites is a common feature of genes (18, 25).

H3K9Ac. Acetylation of lysines eliminates their positive charge which, when it occurs on certain histone tails, has been shown to have a negative effect on the higher order structure of chromatin, essentially making it more open (26). Acetylation of histone H3 lysine 9, H3K9Ac, is associated with actively transcribed genes (26). This lysine is thought to be a substrate for the GCN5/PCAF acetyltransferase (26, 49); although, unlike histone methylation which is mediated by a specific enzyme, acetylation of a given histone lysine may be mediated by multiple enzymes. Figure 2, A and B, third row, shows the kinetic profiles of H3K9Ac in RMC and HTC-IR cells. At the promoter of Lamc1, in both RMC and HTC-IR cell lines there were moderate levels of H3K9Ac which increased less than twofold after 1 h of TPA treatment. In RMC, at the first exon and first few hundred bases of the first intron, the H3K9Ac level increased by nearly threefold after 1 h of TPA treatment, whereas in HTC-IR cells, the levels at these regions remained constant. In both cell types, the levels of H3K9Ac were highest at the 5'-end which is consistent with several studies of histone acetylation patterns of genes (6, 25, 30).

H3K4m2 and H3K4m3. Di- and trimethylation of H3 lysine 4, H3K4m2 and m3, are associated with actively transcribed genes (34, 38, 52). It has been suggested that H4K4m3 marks on open (ON) chromatin state by recruiting ATP-dependent chromatin remodeling complexes (26). In addition, H3K4m3 has recently been shown to help anchor the general transcription factor TFIID to promoters (67). These modifications are mediated by mammalian homologs of the yeast Set1 methylatransferase, a component of the COMPASS complex (17, 52). The fourth and fifth rows of Fig. S1A and S1B show the kinetic profiles of H3K4m2 and m3 at Lamc1 in RMC and HTC-IR cells. In both cell types, H3K4m2 and m3 were highly enriched at the 5'-ends of the Lamc1 locus and changed little (less than 2-fold increase) after treatment with TPA. A low level of H3K4m2 but not H3K4m3 was present at the Lamc1 3'-end in both cell types. These patterns are consistent with studies in yeast and mammals of H3K4 methylation at transcribed genes (4, 7, 18, 25).

While the H3K4m2 level at the promoter in RMC was similar to that of the background seen at ~20 kb upstream of the transcription start site (Fig. S2A), the level at the promoter in HTC-IR cells was four- to eightfold higher than background (Fig. S2B). The Set1 containing COMPASS complex is recruited to the COOH-terminal domain (CTD) of the largest subunit of Pol II (28). Thus, this difference could reflect that the level of Pol II at the Lamc1 promoter in HTC-IR cells is higher (as compared with the background at –20 kb) than in RMC.

H3K36m3. Trimethylation of histone H3 lysine 36, H3K36m3, is a mark associated with transcription elongation in transcribed genes (26, 57). H3K36m3 is thought to function in elongation by recruiting the Rpd3 deacetylase complex and preventing aberrant transcription initiation within the transcribed region of the gene (22). This modification is catalyzed by the Set2 methyltransferase which is associated with the CTD of elongating Pol II (24). The seventh row in Figs. S1A and B shows the kinetic profile of H3K36m3 in RMC and HTC-IR cells. In contrast to H3K4 methylation and H3K9 acetylation profiles in RMC, H3K36m3 levels at the 5'-end of Lamc1 were at their lowest and gradually increased toward the 3'-end of the gene. H3K36m3 was largely unaffected by TPA treatment in these cells. Similarly, in HTC-IR cells, H3K36m3 levels were lowest at the promoter, increased to the 3'-end of the gene, and changed little after TPA treatment. This pattern of Lysine 36 methylation, high levels within the transcribed portion of the gene and little or none at the promoter, is a common feature of transcribed genes (4, 7). The two cell types differed at the first exon of Lamc1 where, compared with the promoter, the level of H3K36m3 was approximately threefold higher in HTC-IR cells than in the RMC.

H3K79m2. Modifications of histone tails are critical for gene expression but modifications of residues within the histone core are thought to play a role in the regulation of chromatin structure (33). H3K79 methylation has been reported throughout euchromatin regions (56) and its involvement in upregulation of Hox genes has been implicated in leukemias (43, 44); however, its function in transcription is unknown. The yeast Dot1p and its human ortholog, hDOT1L, dimethylate lysine 79, H3K79m2 (43). The pattern of H3K79m2 in both RMC and HTC-IR cells was similar with the lowest levels being at the extreme 5'- and 3'-ends and the highest levels observed being at the beginning of the first intron (Fig. S1A and S1B, row 8).

H3K27m3. Trimethylation of lysine 27 of H3, H3K27m3, is a marker of silent genes (34). This modification is mediated by the histone methyltransfease, Ezh2 (13, 48). In agreement with previous studies on H3 lysine 27 methylation (4, 9, 48), the level of H3K27m3 was significantly higher within the 5'-flanking region (–20 kb) of Lamc1 in both cell types than along the promoter and transcribed region (Fig. S1A and S1B, row 9). As expected, H3K27m3 levels were high in the coding region of the silenced β-globin gene locus (data not shown).

RNA Pol II and Histone Changes Associated with TPA Induction of the Short egr-1 Gene

The induction of egr-1 and Lamc1 expression by TPA differs both in the kinetics and amplitude of the increase. Aside from the differences in their promoters, both genes differ greatly in size which could affect the chromatin environment along the genes. We examined the Pol II and histone modification profiles during TPA induction of egr-1 in RMC and HTC-IR cells to see whether they are different from the long gene Lamc1.

Pol II. Consistent with the greater than 10-fold increase in egr-1 mRNA, after 1 h of TPA treatment, Pol II density increased several fold at the promoter and the entire length of the transcribed region of the egr-1 locus (Fig. 3A). The highest peak levels of Pol II presence occurred at the 5'-end of egr-1 while the peak at the 3'-end was no more than twofold lower. This contrasts with Pol II levels at the Lamc1 gene in RMC where peak Pol II levels decrease sixfold from the exon 1 to the 3'-end (exon 28). While the peak Pol II level is highest at the first exon in RMC, it is highest at the promoter in HTC-IR cells. In contrast, the kinetics of hnRNP K recruitment along egr-1 were similar in RMC and HTC-IR cells.

Histone H3. In contrast to the RMC steady levels of histone H3 at the Lamc1 locus (Fig. 2A), there was transient loss of histone H3 at the promoter and transcribed regions of egr-1 which coincided with inducible recruitment of Pol II (Fig. 3A). These observations are consistent with several recent reports of nucleosome depletion associated with gene expression, an effect inversely proportional to the rate of gene transcription (11, 29, 37). Compared with RMC, in HTC-IR cells the H3 levels changed little. Thus, the differences in histone loss reflect not only the level of induction of a gene but also differences in cell type.

Comparison of Pol II and histone modifications at the 5'- and 3'-ends of the Lamc1 and egr-1 genes in RMC. With the exception of H3K9Ac, which exhibited a robust increase at the promoter, the other H3 modification signals in the egr-1 transcribed region showed a transient decrease, which likely reflected total H3 loss (Fig. S2). Changes in nucleosome density along the induced egr-1 locus could conceal changes in histone modifications in response to TPA and between different regions of egr-1. The levels of the H3 modifications at the first and last exons of both egr-1 and Lamc1 were normalized to total H3 and along with Pol II are presented in Fig. 4. The most striking observation was that the differences in the overall levels of Pol II and the histone modifications between the 5'- and 3'-ends of egr-1 were much smaller than for Lamc1. Also, this analysis uncovered an inducible increase in the levels of trimethylated H3K4 and H3K36 at exon 1 of the egr-1 gene which were much smaller at exon 1 of Lamc1 (Figs. S1A and S3). Changes in H3K4m3 and H3K36m3 levels paralleled Pol II recruitment suggesting that these events are related (54).


Figure 4
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  		Fig. 4. Comparison of inducible Pol II recruitment and histone H3 modifications normalized to the amount of histone H3 at the 5'- and 3'-ends of the Lamc1 and egr-1 genes in RMC. RMC were analyzed by ChIP as described in Figs. 2 and 3 with primers to exon 1 and 28 of Lamc1 (A) and exon 1 and 2 of egr-1 (B). Graphs represent IP/Mock IP signal ratios which were normalized to the histone H3 occupancy. Data represent means ± SE from 3 independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We carried out comparative analysis of chromatin states and transcription of the 125-kb-long Lamc1 and 4-kb-long egr-1 genes in RMC and HTC-IR cells. Our data indicate that the length of the Lamc1 locus and also the magnitude of induction determine the profile of the Pol II and histone modification patterns along the gene. In addition, comparison of the induction of Lamc1 in RMC and HTC-IR cells revealed that the difference in Lamc1 mRNA inducibility between the two cell types correlated with a difference in elongation efficiency and histone acetylation along this locus.

Several genome-wide studies have explored constitutive transcriptional and chromatin states in different cell types (4, 14, 18, 20, 25). None of the studies were done in renal cells. We found that in RMC the Pol II and chromatin marks along the Lamc1 and egr-1 genes (Figs. S1A and S2) were similar to the profiles described previously for other constitutively transcribed loci (4, 14, 18, 20, 25). Namely, peak levels of Pol II and histone modifications H3K4m2, H3K4m3, and H3K9Ac were localized to the 5'-end, H3K79m2 in the middle of the gene, and H3K36m3 toward the 3'-end. Major differences between the Lamc1 and egr-1 genes were seen at the 3'-ends, where high levels of H3K4m2, H3K4m3, and H3K9Ac were present at the egr-1 but not at the Lamc1 locus. Because Lamc1 matches the histone profile reported for an average gene (25 kb in mammals) (4, 18, 25), the differences seen here could reflect the short length of egr-1. Histone modifications are thought to affect chromatin structure and transcription by directly changing the affinity of the histone proteins for DNA, and/or by regulating recruitment of accessory factors like ATP-dependent chromatin remodeling complexes and others (26). The resulting changes then can affect the efficiency of Pol II elongation through the nucleosomal array (38, 68). The larger difference in the composition of histone modifications between the 5'- and 3'-ends, seen here in the long Lamc1 gene, could result in a larger difference in Pol II elongation efficiency between the two ends. To our knowledge, no studies have been done on the differences in transcription elongation between short and long genes and our data suggest that length of a gene is a factor regulating Pol II elongation.

In comparing Lamc1 transcription in two cell types, we found that Pol II recruitment to the Lamc1 promoter is increased in HTC-IR cells treated with TPA; however, there is no Pol II increase downstream of the promoter and no changes in the mRNA level were detected. We suggest that in HTC-IR cells the inducibly recruited Pol II either forms an unstable initiation complex or is not competent to transition to productive elongation and instead, eventually falls off the DNA. Furthermore, comparison of Pol II profiles reveals similarities between the two different genes, Lamc1 and egr-1, in HTC-IR cells (Figs. 2B and 3B). The highest overall level of Pol II at the Lamc1 locus and the highest peak level at the egr-1 locus are at the promoter. Accumulation of Pol II at the promoter suggests that there is a limiting step immediately downstream, indicating that the slowest step in Pol II transcription of the Lamc1 and egr-1 genes in HTC-IR cells is postinitiation, at a very early stage of elongation (e.g., promoter clearance). It is possible that in HTC-IR cells, the concentration or activity of an early elongation factor is below the threshold required for Lamc1 induction by TPA, therefore initiation by the Pol II complex at the promoter is nonproductive. In contrast, in RMC cells, Pol II accumulates further downstream at exon 1 of Lamc1 and the highest peak level at egr-1 is also within the first exon, indicating that, in these cells, the slowest step in transcription resides within the coding region of genes.

Several factors are required for Pol II to clear the promoter, including TFIIH, F, and S, P-TEFb, DSIF, and Paf1, providing a number of possible means for regulating transcription at early steps of elongation (53). It has been suggested that regulation of gene expression at a point immediately after Pol II and the general transcription factors form the preinitiation complex that is fairly common (23) but despite this, relatively few examples of the regulation of gene expression at this level have been elucidated (51, 60).

Another factor that may regulate early steps of elongation is the acetylation of histones. It is known that gene transcription is associated with histone acetylation (including H3K9Ac) at 5'-end and the amount of acetylation appears to be important to the activation of the gene (6, 25, 27). Aside from its function in transcription initiation, histone acetylation may regulate the release of Pol II from the promoter (10). H3K9Ac downstream of the Lamc1 promoter is increased in response to TPA in RMC but not the HTC-IR cells. It is therefore possible that a nucleosomal block to elongation exists downstream of the Lamc1 promoter and that elongation by inducibly recruited Pol II requires concomitant acetylation of histones to relieve this block. Taken together, comparative analysis in the two cell types suggests that the transcriptional control steps of a given gene are not the same for all cells.

Previous studies have highlighted the importance of correct Lamc1 expression in the development and function of tissues (40, 59). For instance, increased expression of the Lamc1 locus is associated with the increased ECM production in diabetic glomerulosclerosis (15). While most studies focused on Lamc1 transcription initiation or posttranscriptional control in renal cells, regulation of Lamc1 elongation in these cells was not examined. Our work identified posttranscription initiation steps as a possible means of regulating Lamc1 expression. To our knowledge, this is the first example of this type of transcriptional control for a gene that encodes a component of the ECM. The approaches we used may help to identify factors that regulate the transcription elongation of Lamc1 and shed light on the mechanisms that shape the specificity of the cell's response to extracellular stimuli.


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This work was supported by National Institutes of Health Grants DK-45978 and GM-45134 (to K. Bomsztyk).


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Bomsztyk, UW Medicine at Lake Union, Box 358050, Univ. of Washington, Seattle, WA 98109 (e-mail: karolb{at}u.washington.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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