extfieldsofstudy: Biology Medicine

text:
Comparison of standard exponential and linear techniques to amplify small cDNA samples for microarrays

Background The need to perform microarray experiments with small amounts of tissue has led to the development of several protocols for amplifying the target transcripts. The use of different amplification protocols could affect the comparability of microarray experiments. Results Here we compare expression data from Pinus taeda cDNA microarrays using transcripts amplified either exponentially by PCR or linearly by T7 transcription. The amplified transcripts vary significantly in estimated length, GC content and expression depending on amplification technique. Amplification by T7 RNA polymerase gives transcripts with a greater range of lengths, greater estimated mean length, and greater variation of expression levels, but lower average GC content, than those from PCR amplification. For genes with significantly higher expression after T7 transcription than after PCR, the transcripts were 27% longer and had about 2 percentage units lower GC content. The correlation of expression intensities between technical repeats was high for both methods (R2 = 0.98) whereas the correlation of expression intensities using the different methods was considerably lower (R2 = 0.52). Correlation of expression intensities between amplified and unamplified transcripts were intermediate (R2 = 0.68–0.77). Conclusion Amplification with T7 transcription better reflects the variation of the unamplified transcriptome than PCR based methods owing to the better representation of long transcripts. If transcripts of particular interest are known to have high GC content and are of limited length, however, PCR-based methods may be preferable.

Background
The analysis of transcript abundance in samples of total RNA using standard techniques such as northern blotting or microarrays requires microgram quantities of total RNA. In our experience, a microarray analysis incorporat-ing a loop design and reciprocal labeling with Cy™3 and Cy™5 dyes, requires 80 micrograms of total RNA per sample [1]. It is often inconvenient or impossible to obtain sufficient quantities without an amplification step, particularly if tissue sections are to be analyzed. Exponential amplification of cDNA by a standard PCR procedure [2] may result in the differential amplification of particular transcripts, since sequences differ in the rate with which they can be amplified by PCR [3]. To minimize this problem, the sequences to be amplified can be limited to about 300 nucleotides at the 3'-terminus of the cDNA; this can be achieved by ultrasound treatment [4] or by limiting the concentration of deoxynucleotides in the PCR reaction mixture [5]. These methods are promising but not yet in standard use. An alternative approach is linear amplification by in vitro transcription from a strong promoter such as a T7 phage promoter [6]. Linear amplification has been shown to retain the relative frequencies of transcripts with reasonable fidelity over a wide amplification range [7][8][9][10]. Many aspects of the high efficiency and reliability of linear and exponential amplification methods have been studied earlier. These deal mainly with comparisons between unamplified and amplified material and indirectly between different amplification methods [11,12]. The distorting effect in mRNA abundance of linear and exponential amplification techniques in relation to the sequence and GC content of the genes has been hypothesized [12], but very little evidence has been put forward to support this in relation to the characteristics of individual transcripts.
Commercial kits are available for both exponential (Super SMART™ from BD Biosciences Clontech) and linear (Message Amp™ from Ambion) amplification. Here we compare expression levels determined with cDNA microarrays hybridized with cDNA obtained from thin sections of secondary xylem tissue from Pinus taeda amplified by these two different strategies, or after using unamplified cDNA for hybridization. We have addressed the questions: how well do the results agree with each other in a direct comparison? What are the characteristics of the sequences showing preferential amplification by exponential or linear amplification?

Comparison of unamplified-and amplified targets
A typical sample for amplified Super SMART™ PCR-product yields a distribution of sizes from 500 bp-6000 bp with a peak centered at 900 bp (Clontech, Super Smart PCR cDNA Synthesis Kit User manual). A typical sample for amplified Message Amp™ aRNA yields a distribution of sizes from 250 nt-5500 nt with a peak centered at 1000-1500 nt (Ambion, Catalog #1752). The distributions of our amplified material agree well with the manufacturers' data (See Additional file 1 and 2). It has been reported that PCR amplification requires less RNA, is more reproducible and generates better target transcripts than linear amplification [5,13], at least if the sequences are limited to the 3'-end. Linear T7 amplification has however been widely used when starting material is limit-ing. Recently some researchers have reported bias in their data. In some studies the bias is said to be of minor importance, systematic and reproducible, affecting all the samples in the same way and therefore potentially controllable in the normalization (e.g. to calculate fold change) [10,14]. In other studies the bias from different amplification protocol is affecting the general ratios of gene expression [5,12]. Part of the bias may arise from the T7 RNA polymerase's intrinsic nucleolytic activity that appears during extended incubation [15]. Other bias is maybe introduced owing to the characteristics of the individual transcripts.
We have found a preferential amplification of certain nucleotide sequences by the Super SMART™ PCR relative to a nonamplified target in earlier membrane array experiments, where the targets were prepared from the samples of lignified planings and nonlignified xylem scrapings (data not shown). The correlation (R 2 ) between transcript abundance using unamplified and Super SMART™ PCR amplified targets was 0.77 for scrapings and 0.68 for planings.
Comparing five lines of Picea abies shoots where the first biological replicate consisted of unamplified targets and the second biological replicate consisted of targets amplified with T7 transcription we obtained a correlation of R 2 = 0.74 (data not shown). Ambion has reported R 2 = 0.87 [16] between technical repeats.
Plots of the individual gene transcript abundance of unamplified versus amplified target should give a straight line of slope 1 if the overall expression is preserved. However, there is some nonlinear behavior in both cases. For unamplified versus PCR amplified target the curve is generally nonlinear and lower abundance transcripts are under-represented and highly expressed transcripts are amplified better than average. For the unamplified versus T7 amplified targets a very small minority of highly expressed transcripts do not follow the linear slope of around 1.
For the highly significant (p < 0.0001) differentially expressed genes between lines in each of the ten comparisons of unamplified and T7 amplified targets, the 95% confidence intervals for the fold-changes were as follows. For unamplified material: Downregulation, 1.5-3.2 (all), and 2.7-7.5 (top); upregulation: 1.3-2.8 (all), and 2.6-4.4 (top). For T7-amplified material: Downregulation, 1.0-2.7 (all), and 2.5-4.2 (top); upregulation: 1.2-3.0 (all), and 2.3-4.3 (top). The differences between unamplified and T7 amplified targets are generally not statistically significant although the fold change for the unamplified targets were greater than for the T7 amplified targets indicating that some small bias may still exist when using T7 amplified relative to unamplified targets, especially for highly expressed transcripts.
However, in many situations there is no possibilty of using unamplified targets and amplification is required. Thus, starting with small amounts of secondary xylem tissue we compared PCR and T7 RNA polymerase amplification methods directly to investigate if, and how, the biases differ from each other.

Expression characteristics of transcripts amplified by PCR or T7 transcription
The two methods of amplification were compared to each other four times and twice to themselves in a fully balanced flip dye experimental design including technical repeats ( Figure 1A). Only few spots were flagged as bad and excluded from further analysis. The percentage of detectable spots (above background) on each array and in each channel was 88% using T7 amplification and 71% using PCR amplification. The percentage of saturated spots was around 1% in all cases.
After normalization the correlation of transcript abundance for each gene between technical repeats was very high, R 2 = 0.98, after both PCR-( Figure 1B) and T7 amplification ( Figure 1C). In contrast, the correlation between the two different amplification methods for both technical repeats was considerably lower, R 2 = 0.52, ( Figure 1D), indicating bias in one or both amplification techniques. As previously mentioned the correlation between unamplified and amplified transcript abundance was intermediate, indicating that both amplification methods have bias and that these biases are different from each other.
The genes present on the microarray were divided into two groups according to whether the PCR amplified transcripts (S') or the T7 amplified transcripts (M') were more abundant. The S' group was 9% larger than the M' group.
A relative frequency distribution plot of expression levels revealed a narrower peak for S' than for M' transcripts (Figure 2A). The arithmetic expression values showed a significantly greater mean for M' (1.76) than for S' (1.64) and a higher variance although the coefficient of variation was lower for M' (81.6%) than for S' (86.6%). The distribution of the data implies a broader population of transcript species present in the T7 amplified target.
Using the criteria for statistical significance described in methods, 309 ESTs (14%) showed different expression levels between the two amplification methods with 131 ESTs in the S' group and 178 ESTs in the M' group. The arithmetic mean of the S' group (3.40) was statistically higher than the M' group (2.95) and the S' group had higher variance ( Figure 2B). The coefficient of variation was lower for M' (33.4%) than for S' (36.3%). The reason for the opposite trend observed for this subset of genes may reflect the differences in detectable spots and the amplification kinetics between PCR and T7 transcription.

Transcript characteristics amplified by PCR or T7 transcription
As shown above, out of the genes (309 ESTs) showing statistically significant abundance differences between the amplification methods, 36% more were found in the M' group than in the S' group. One possibility for why 36% more were found in the M' group is that the complexity of the T7 amplified transcripts is greater. To assess this we analyzed the length of the sequences on the array. Previous analyses of protein sequences showed about half of Pinus taeda ESTs on the array have an apparent homolog in Arabidopsis thaliana (increasing with length up to 90%). For these ESTs the sequence similarity is typically distributed over the full length of the contig indicating a substantial conservation of genes between these two species, suggesting a common functional genome [17]. From the BLASTn™ (nucleotide level) and BLASTx™ (amino acid level) searches relating the contig data to Arabidopsis thaliana homologs, the corresponding Pinus taeda full-length cDNAs were estimated. The contig lengths constitute on average about 45 % of the total cDNA lengths spotted on the array. For both the nucleotide and the amino acid levels there was a highly significant 60% greater variance in length of the M' group than of the S' group. At the amino acid level there was a significant 26.9% greater mean length of the M' group (1580 bp) than the S' counterpart (1245 bp). The maximum length of transcript present was also considerably greater in the M' group than the S' group ( Figure 2C). In contrast to the contigs the singleton ESTs in the S' group (482 bp) had a significantly greater mean sequence length than those in the M' group (428 bp). The reason for this discrepancy is unclear but could reflect a difference in efficiency of the sequencing polymerase resulting from difference in the amount of secondary structures in the sequences from the two sets. The M' group contained 60% of the ESTs and contigs with nucleotide and amino acid homology to Arabidopsis thaliana reflecting both an initially greater transcript population as well as differences in transcript lengths. In conclusion, the possibility of getting transcripts of greater length and larger variability is considerably higher when using T7 amplification rather than PCR amplification.

Importance of GC content for amplification
Comparison of the selected genes (309 ESTs) differentially represented in the two amplification methods, the GC content of the ESTs, contigs and Arabidopsis thaliana cDNAs (on a nucleotide level) there was a significantly greater mean GC content for the sequences of the S' group than for those of the M' group. The difference was 2.7 percentage units for ESTs, and 1.4 percentage units for the corresponding contigs ( Figure 2D). There was a similar difference for the cDNAs although only about 10% of the contigs were found to have a BLASTn™ score above 100 bits. Interestingly, for a smaller group of 80 contigs (40 from S' and 40 from M') showing the greatest fold changes between methods, the difference in GC content increased from 1.4 to 2.2 percentage units, due to an increase in GC content for the S' group. Additionally, the mean length of the 40 ESTs from the S' group (1428 bp) was significantly greater than the mean length of the 40 ESTs from the M' group (1275 bp). It appears that transcripts with a high GC content are amplified faster by PCR than by T7, often overriding the effect of length. If the GC content is nearer the average, long transcripts are favored by T7 amplification. The GC effect is presumably explained by the temperature of extension, which is 68-72°C for Taq polymerase and 37°C for T7 polymerase; high temperature favors polymerization through GC-rich areas. Evolution has in general tuned the cellular machinery, including polymerases, to fit the temperature environment of an organism. This might be reflected in the GC content and the temperature environment of the original organism for each polymerase. The GC content of a Pinus species genome is about 40%, which is considerably closer to the 48% GC content of T7 phage (or the 50% GC content of Escherichia coli, the typical host of T7 phage), than for the 67% GC content of Thermus aquaticus [18][19][20]. It implies that T7 transcription of the Pinus taeda transcrip- tome or consequently other transcriptomes with similar GC content in most cases is a better choice than PCR based techniques.

Conclusion
In summary, the two main approaches to amplification of small amounts of RNA for microarray studies, PCR and T7 transcription both introduce bias compared to the unamplified target and the nature of the bias is different for each method. Our results show that amplification by T7 RNA polymerase gives transcripts with a greater range of lengths, greater estimated mean length, and greater variation of expression levels, but lower average GC content, than those from PCR amplification. Amplification with T7 transcription would therefore better reflect the variation of the unamplified Pinus taeda transcriptome and other comparable transcriptomes than PCR based methods. If transcripts of particular interest are known to have high GC content and are of limited size, however, PCR based methods may be preferable. The results demonstrate the need to pay attention to possible biases introduced by the amplification methods and that in certain projects different amplification techniques should be tested and optimized before routine use.

Target extraction and amplification
Polyadenylated RNA was extracted from individual 30 µm cryotome sections (3 mm × 3 mm) through the cambial region of Pinus taeda L. using Dynabeads (Dynal Biotech, Oslo, Norway). The mRNA samples were reverse-transcribed and the resulting cDNA were amplified by a) exponential PCR amplification by Super SMART™ (BD Biosciences Clontech, Palo Alto, CA, USA), or b) linear T7 amplification through Message Amp™ aRNA kit (Ambion, Austin, TX, USA). The Super SMART™ cDNA products were directly labeled by Klenow with Cy™3 or Cy™5 dUTPs (Amersham Biosciences, Piscataway, NJ, USA). The Message Amp™ aRNA products were reverse-transcribed with aminoallyl-modified dUTPs (Sigma, St. Louis, MO, USA) and labeled by coupling to free Cy™3 or Cy™5 dye (Amersham Biosciences) ( Table 1).

Microarray hybridization and probe selection
Microarray hybridization and stringency washes have been described previously [21,22]. cDNA microarrays based on 2190 Pinus taeda ESTs from the NSF unigene set (Forest Biotechnology Group, NCSU, NC, USA) [17] were hybridized with the labeled targets. The PCR and T7 amplification methods were compared in a fully bal-  Figure 1A). The microarray data is MIAME compliant [GEO:GPL1880].

Data normalization and analysis
The consistency of each method was assessed by dividing the samples into two technical repeats. The slides were scanned using a ScanArray ® 4000 Microarray Analysis System (GSI Lumonics, Ottawa, Canada). Raw intensity values were collected with QuantArray ® software (GSI Lumonics) and spots were visually inspected for spot morphology and background. No background subtraction was applied because backgrounds were low and subtraction can introduce bias. The microarray intensity data was normalized using a mixed model system [21,[23][24][25] in SAS/STAT Software version 8 (SAS Institute Inc., Cary, NC, USA). The log 2 fold change in abundance was used to divide the selected genes in two groups depending on sign.
The normalized log 2 fold change (essentially a ratio of the least square means of Super SMART™-and Message Amp™-amplified transcript abundance derived from the mixed model) with a probability value of p < 0.001 and array-and array*dye interaction variance lower than 0.001 were used to select genes with significant changes in abundance (represented by 309 ESTs). The absolute values (i.e. a rescaling of the data disregarding the sign) of the log 2 fold change abundance were then used in the subse- The lengths of the cDNAs represented on the microarray were estimated based on full length Arabidopsis thaliana [26] homolog sequences using the Pinus taeda ESTs and contigs [27]. The top Arabidopsis thaliana homolog cDNAs with a score greater than 100 bits were selected for Pinus taeda ESTs or contigs on nucleotide level (using BLASTn™ and the AGI transcripts (-introns, +UTRs) dataset) or amino acid level (using BLASTx™ and the AGI proteins dataset).
All the Pinus taeda ESTs and contigs including those subsets showing homology to Arabidopsis thaliana cDNAs were then analyzed for sequence length, GC content as well as log 2 fold change abundance.
The corresponding groups in each subset were analyzed with Prism Software version 3 (GraphPad Software Inc., San Diego, CA, USA). F-tests were used for evaluating a group's compliance with Gaussian distribution. When the normal criteria were met for two groups, one-way ANOVA analysis (with Bonferroni post test) and unpaired t-tests with or without applicable Welch's correction (not assuming equal variances) were performed. When the normal criteria were not met the nonparametric Mann-Whitney test was performed.


GLUT4 ready to go

Vesicles containing the glucose transporter GLUT4 travel rapidly on a microtubule network that lies just under the plasma membrane in resting cells, according to results from Lizunov et al. (page 481). The vesicles occasionally touch the plasma membrane and, when stimulated by insulin, quickly fuse with it.

Figure

Vesicles with GLUT4 move along microtubules near the plasma membrane.

GLUT4 is sequestered in vesicles in resting cells. Insulin exposure induces the vesicles to fuse to the plasma membrane. But how these vesicles are stored in the resting cell was unclear.

To find out, the team used total internal reflection fluorescence (TIRF) microscopy on freshly isolated adipose cells transfected with GLUT4-GFP. Whereas confocal microscopy illuminated vesicles scattered throughout the cytoplasm, TIRF movies showed the vesicles just under the plasma membrane. These sub-membrane vesicles were moving on microtubules in unstimulated cells. Within ten minutes of insulin exposure, 50% of the GLUT4 had reached the surface—and most of the vesicles on the microtubule network were clustered at sites on the membrane.

The authors aim to find out what the vesicles are doing as they move around the resting cell, sampling the plasma membrane. They speculate that the moving vesicles might allow the cell to rapidly respond to a narrow area of insulin exposure.

GLUT4 ready to go
esicles containing the glucose transporter GLUT4 travel rapidly on a microtubule network that lies just under the plasma membrane in resting cells, according to results from Lizunov et al. (page 481). The vesicles occasionally touch the plasma membrane and, when stimulated by insulin, quickly fuse with it.
GLUT4 is sequestered in vesicles in resting cells. Insulin exposure induces the vesicles to fuse to the plasma membrane. But how these vesicles are stored in the resting cell was unclear.
To find out, the team used total internal reflection fluorescence (TIRF) microscopy on freshly isolated adipose cells transfected with GLUT4-GFP. Whereas confocal microscopy illuminated vesicles scattered throughout the cytoplasm, TIRF movies showed the vesicles just under the plasma mem-V brane. These sub-membrane vesicles were moving on microtubules in unstimulated cells. Within ten minutes of insulin exposure, 50% of the GLUT4 had reached the surfaceand most of the vesicles on the microtubule network were clustered at sites on the membrane.
The authors aim to find out what the vesicles are doing as they move around the resting cell, sampling the plasma membrane. They speculate that the moving vesicles might allow the cell to rapidly respond to a narrow area of insulin exposure.
Vesicles with GLUT4 move along microtubules near the plasma membrane.

Evidence for AMPAR association
haracterization of AMPA receptors (AMPARs) purified from wild-type mice and from a mouse strain carrying an epitope-tagged version of the GluR2 subunit shows that AMPARs interact with only a subset of proteins previously thought to associate with the receptors, report Fukata et al. (page 399).
Changes in the number of AMPARs alter how well synapses conduct currents in the brain and are thought to control memory storage. A large number of proteins have been found to associate with AMPARs by in vitro assays, such as two-hybrid screens or coexpression in cell lines, but evidence for functional interaction is limited.
When Fukata et al. purified AMPAR components from brain extracts of transgenic mice expressing the epitope-tagged GluR2, only BiP, a common ER chaperone, and transmembrane AMPAR regulatory proteins (TARPs) came down in a quantitative manner. Because BiP and TARPs were in distinct AMPAR complexes, the researchers hypothesized that BiP-GluR2 may be an immature transitory complex.
Why didn't the other known AMPAR proteins come down? The authors think that only a few of the previously identified proteins associate with the majority of AMPARs in brain cells. The other proteins might interact with the receptors in a small subset of cells or under specific conditions. And the new work is consistent with emerging genetic data showing that, when these other proteins are removed, there are limited effects as compared with the phenotype of the stargazer mouse, a TARP mutant that lacks functional AMPARs in a common neural cell type in the brain. Claudin-19 fit the bill. Mice lacking functional claudin-19 showed behavioral and morphological abnormalities suggestive of peripheral nerve damage and disruption of the tight junction-like structures. Recordings of compound action potentials showed two currents, one fast and one slow. This change in conductance properties indicated that, without claudin-19, the myelin sheath of Schwann cell appeared to be inadequately sealed from the environment. Thus the mutation may have interfered with normal saltatory travel of neural currents.
The dependence on tight junctions may be greater in thinner myelinated axons than in thicker ones because the thinner axons lack the bulk that would compensate for improperly sealed wrappings. The team is continuing to follow the now two-year-old mice to see if a lack of proper tight junctions induces demyelination later in life.


extfieldsofstudy: Medicine Biology

text:
Microspherule Protein 1, Mi-2β, and RET Finger Protein Associate in the Nucleolus and Up-regulate Ribosomal Gene Transcription*

The nucleolus is the site of ribosomal DNA (rDNA) transcription and ribosome production. In exploring the role of nucleolar protein MCRS1 (microspherule protein1)/MSP58 (58-kDa microspherule protein), we found that Mi-2β, a component of a nucleosome remodeling and deacetylase (NuRD) complex, RET finger protein (RFP), and upstream binding factor (UBF) were associated with MCRS1. Yeast two-hybrid assays revealed that MCRS1 bound to the ATPase/helicase region of Mi-2β and the coiled-coil region of RFP. Interestingly, confocal microscopic analyses revealed the co-localization of MCRS1, Mi-2β, RFP, and the rRNA transcription factor UBF in the nucleoli. We also found that MCRS1, Mi-2β, and RFP were associated with rDNA using a chromatin immunoprecipitation assay. Finally, we showed that MCRS1, Mi-2β, and RFP up-regulated transcriptional activity of the rDNA promoter and that ribosomal RNA transcription was repressed when MCRS1, Mi-2β, and RFP expression was reduced using siRNA. These results indicated that Mi-2β and RFP, known to be involved in transcriptional repression in the nucleus, co-localize with MCRS1 in the nucleolus and appear to activate the rRNA transcription.

The nucleolus is a subnuclear compartment in eukaryotic cells responsible for rRNA synthesis and ribosome biogenesis. In actively growing animal and plant cells, rRNA synthesis accounts for about 50 -80% of total cellular RNA production (1). Human diploid cells contain about 400 ribosomal genes organized as tandem repeats at nucleolus organizer regions encoded on chromosomes 13, 14, 15, 21, and 22 (1,2). rRNA gene transcription varies according to the demand for ribosome production and protein synthesis, and in yeast and murine cells, only 30 -50% of the rRNA genes are in an open structure that facilitates active transcription (3,4). Transcription of rRNA is extensively regulated by a large number of proteins such as growth factors, CBP (cAMPresponse element-binding protein (CREB)-binding protein), Rb, p53, and Myc (5)(6)(7)(8)(9)(10). In addition, growing evidence indicates roles for different histone modification and chromatin remodeling complexes in the coordinated regulation of rRNA gene transcription. Nucleolar remodeling complex (NoRC) is a well recognized chromatin remodeling complex involved in rRNA gene silencing and is known to cooperate with DNA methyltransferases, histone deacetylases, histone methyltransferases, and TTF-I (transcription terminator factor) (11)(12)(13)(14). In contrast, ribosomal DNA transactivation is associated with Tip60 histone acetyltransferase and histone acetylation (15,16). However, the chromatin remodeling proteins that facilitate the establishment of the open state in rRNA genes remain to be characterized.
Mi-2 is a nuclear protein with chromatin remodeling activity and is the largest component of the nucleosome remodeling and deacetylase (NuRD) 3 complex (17)(18)(19)(20). The transcriptional repressive activity of Mi-2 is mediated by NuRD complex components, such as methyl CpGbinding protein MBD 2/3, histone deacetylases HDAC1/2, metastasisassociated protein MTA2/3, and RbAp46/48. NuRD complex and Mi-2 have been associated with the transcriptional repressive activities of KAP-1, RET finger protein (RFP), Trk69 (Tram-track69), and ROR␥ (21)(22)(23)(24). In addition, MTA-3, another component of the NuRD complex, was reported to repress Snail expression, and the estrogen-dependent transcriptional control of MTA3, Snail, and E-cadherin was associated with breast cancer progression (25). However, Mi-2 also plays roles in transactivation outside the NuRD complex. A transactivating role of dMi-2 was suggested in studies of Drosophila salivary glands that showed the localization of dMi-2 to chromatin puffs (26). We recently reported that Mi-2␤ possessed both transactivating and repressing subdomains (24). Furthermore, the study, based on a conditional Mi-2␤ knock-out mouse line, clearly showed that Mi-2␤ activated CD4 transcription in T cells by forming a complex with p300 histone acetyltransferase and the E box-binding protein HEB (27). Thus, Mi-2␤ appears to be involved in both transcriptional repression and activation through the formation of distinct protein complexes in the nucleus.
The RFP was originally identified as a fusion protein with RET-tyrosine kinase (28). RFP mRNA is strongly expressed in a variety of human and rodent tumor cell lines. In addition, its protein expression is detected strongly in male germ cells and relatively weak in peripheral and central neurons, hepatocytes, and adrenal chromaffin cells (28,29). RFP contains a tripartite motif consisting of a RING finger, a B-box zinc finger, a coiled-coil domain that is involved in protein-protein associations, and an RFP domain. RFP is a nuclear protein and was reported to partially co-localize with promyelocytic leukemia protein and int-6 in the nucleus (30,31). We found that RFP exhibited transcriptional * This work was supported by grants-in-aid for the 21st Century Center of Excellence repressive activity and that it was associated and co-localized with Enhancer of Polycomb 1 and Mi-2␤ in the nucleus (24,32). We also showed that the subnuclear distribution of RFP was altered by small ubiquitin-like modifier modification by protein inhibitor of activated STAT (signal transducers and activators of transcription) (PIAS) proteins (33). MCRS1 (microspherule protein 1)/MSP58 (58-kDa microspherule protein) and the related protein p78 were originally identified as proteins that interacted with nucleolar protein p120 and herpes simplex virus 1-infected cell protein 22 (ICP22) (34,35). MCRS1 is a nucleolar protein that contains a bipartite nuclear localization motif, a nucleolar localization motif, a coiled-coil domain, and a forkhead associated domain. Several functions for MCRS1 and its splice variant MCRS2 have been reported, including a transforming activity, nucleolar sequestration activity, and telomerase inhibition (36 -39). MCRS1 and TOJ3, a protein with high structural similarity to MCRS1, exhibit transforming activity, whereas phosphatase and tensin homologue (PTEN) suppresses the transforming activity of MCRS1 (36,38). It has also been shown that MCRS1 can relieve the repressor activity of Daxx, an adaptor protein that links Fas signaling to the c-Jun NH 2 -terminal kinase pathway via a nucleolar sequestration mechanism (39). MCRS2 is involved in telomere shortening by associating with telomerase (37). In addition, binding and stabilization of MSP58 by transcription factor STRA13 has been observed (40). Although these findings suggest that MCRS1 may have several distinct functions, its transcriptional role in the nucleolus has not yet been fully characterized.
In this report we investigated the role of MCRS1 in ribosomal gene transcription and its association and co-localization with Mi-2␤ and RFP in the nucleolus. Our results indicated that Mi-2␤, RFP, and nucleolar protein MCRS1 were involved in ribosomal gene transcription and suggested that Mi-2 may be a candidate for the unidentified chromatin remodeling protein that establishes the euchromatin structure of ribosomal genes.

MATERIALS AND METHODS
Plasmids-MCRS1/MSP58 cDNA (GenBank TM accession number NM_006337) was obtained by PCR using a human testis cDNA library as a template and sequenced. The same product was obtained using a human placenta cDNA library as a template. The full-length MCRS1 was cloned in-frame into pEGFP-C1 plasmid (Clontech).
Human ribosomal DNA promoter spanning Ϫ483 to ϩ377 bp with respect to the transcription initiation site (GenBank TM accession number U13369) (41) was amplified from genomic DNA isolated from human embryo kidney (HEK) 293 cells and checked by sequencing. The internal ribosome entry site (IRES) from the encephalomyocarditis virus was amplified from pIRES2-EGFP vector (Clontech) and checked by sequencing. The Kozak sequence of the pGL3-basic vector was removed by digestion with NcoI and HindIII and replaced with the IRES sequence to generate the pGL3-IRES-Luc plasmid (42). The pGL3-GAL4-hrDNAP-IRES-Luc plasmid was generated by cloning the GAL4 DNA binding region (GAL4BD) fragment of pAS2-1 vector (Clontech) and the human ribosomal DNA promoter (Ϫ483 to ϩ377) into the pGL3-IRES-Luc plasmid. The pcDNA3-GAL4BD plasmid was described previously (32), and the nucleolar localization signal (NoLS) of MCRS1 (32-57 amino acids) was inserted right after the GAL4BD sequence to generate the pcDNA3-GAL4BD-NoLS plasmid.
Antibodies-Anti-RFP polyclonal antibody was described previously (24,32). Anti-Mi-2 polyclonal antibody was a kind gift from Dr. Wade at Emory University (43). Anti-MCRS1 polyclonal antibody was developed against the 19 carboxyl-terminal amino acids of MCRS1 (Gen-Bank TM accession number NM_006337). Anti-FLAG (M2) monoclonal antibody was obtained from Sigma. Anti-upstream binding factor (UBF) monoclonal antibody was purchased from Santa Cruz Biotechnology. Anti-GFP polyclonal antibody was purchased from MBL (Japan). Alexa-conjugated secondary antibodies were purchased from Molecular Probes. Anti-MCRS1 polyclonal antibody was labeled with Alexa 594 using the Alexa Fluor 594 protein labeling kit (Molecular Probes).
Western Blotting and Co-immunoprecipitation Experiments-HEK 293 cells plated in 100-mm dishes were transfected with 4 g of pFLAG-CMV2 expression vector or pEGFP-C1-MCRS1 expression vector using Lipofectamine PLUS (Invitrogen) according to the manufacturer's instructions. After 48 h of incubation, cells were washed twice with ice-cold washing buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl) and resuspended in 1 ml of hypotonic buffer (10 mM Tris-HCl, pH 7.5, 5 mM MgCl 2 , and 1 mM phenylmethylsulfonyl fluoride (PMSF)) containing Complete protease inhibitor mixture (Roche Diagnostics). The suspension was briefly sonicated and mixed with 160 l of 2.5 M sucrose solution. After centrifugation at 1200 ϫ g for 5 min at 4°C, the pellet was lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 20 mM MgCl 2 , 0.5% Nonidet P-40, and 1 mM PMSF) containing Complete protease inhibitor mixture and centrifuged again at 12,000 ϫ g for 25 min at 4°C. The resultant supernatants were incubated with specific polyclonal antibodies or mouse anti-FLAG M2 antibody coupled with protein A-Sepharose or protein G-Sepharose (Sigma) for 5 h at 4°C. Coimmunoprecipitation, electrophoretic separation, and Western blotting were performed as previously described (24). The nuclear/cytosol fractionation kit (Bio Vision) was used to prepare nuclear extracts to check the specificity of anti-MCRS1 antibody.
Yeast Two-hybrid Assay-The SfiI/PmeI fragment of the pcDNAV5-HisC-HA Mi-2␤ full was cloned in-flame into the SfiI/SmaI site of the pAS2-1 vector (Clontech), and fragments of Mi-2␤ were cloned into the SfiI/SalI site of the pAS2-1 vector (24). pAS2-1-RFP constructs were as described previously (32). Fragments of MCRS1 were amplified by PCR and cloned into the pACT2 vector (Clontech). To characterize regions of Mi-2␤ and RFP that mediated binding to MCRS1, pAS2-1-Mi-2␤ or pAS2-1RFP constructs were co-transfected with the pACT2-MCRS1 vector into S. cerevisiae strain Y190 (Clontech). Positive interactions were determined by two criteria, expression of ␤-galactosidase and growth on the triple drop-out medium without tryptophan, leucine, and histidine. Measurements were performed on at least three independent colonies.
GAL4-fused Ribosomal DNA Promoter-targeted Transcription Assay-Mouse L cells were kindly provided by Dr. Kaibuchi at Nagoya University. HEK 293 cells and mouse L cells were cultured in 24-well tissue culture plates and co-transfected with 40 ng of GAL4-hrDNA promoter-IRES luciferase reporter plasmid, 40 ng of pRL-TK plasmid (Promega), and 320 ng of pcDNA3-GAL4BD Mi-2␤, RFP, or MCRS1 expression plasmids with or without NoLS using Lipofectamine PLUS (Invitrogen). Cells were harvested 48 h after transfection, and luciferase assays were performed as previously described (32). Co-transfection with the pRL-TK plasmid was used to normalize luciferase values. Values were expressed as the means Ϯ S.D. of at least three independent experiments.
RNA-mediated Interference -MCRS1 and Mi-2␤ siRNA and control siRNA (siControl non-targeting siRNA) were purchased from Dhama-con, and RFP siRNA was purchased from Qiagen. The siRNAs were transfected using X-tremeGENE siRNA transfection reagent (Roche Applied Science) according to the manufacturer's instructions.
Ribosomal RNA Expression Analysis-Total cellular RNA was isolated using the RNeasy Mini kit (Qiagen), and residual DNA was digested using RNase-free DNase (Qiagen). RNA was reverse-transcribed with MultiScribe reverse transcriptase (Applied Biosystems) for 30 min at 48°C. The resultant cDNA was amplified by PCR for 12-40 cycles of 30 s at 96°C, 30 s at 55°C for 28 S ribosomal RNA and 47 S/45 S ribosomal RNA or 65°C for GAPDH, and 30 s at 72°C. PCR primer sets used for 47 S/45 S ribosomal RNA were forward primer, 5Ј-CGAA-GAAGCGTCGCGGGTCT-3Ј, and reverse primer, 5Ј-CCAAGTAG-GAGAGGAGCGAGC-3Ј, and primer sets for 28 S ribosomal RNA and GAPDH were the same as those used for the ChIP assay.

MCRS1 Interacts with Mi-2 and RFP-MCRS1
is a nucleolar protein that contains a bipartite nuclear localization motif, a nucleolar localization motif, a coiled-coil domain, and a forkhead-associated domain. MCRS1 is involved in the nucleolar sequestration of Daxx and was recently reported to have transforming activity (38,39). In a survey of MCRS1-interacting proteins by immunoprecipitation of lysates from HEK 293 cells transfected with FLAG-MCRS1 (Fig. 1A), we repeatedly identified a 220-kDa protein that co-precipitated with FLAG-MCRS1. Because 220 kDa was consistent with the molecular mass of Mi-2 and  NOVEMBER 25, 2005 • VOLUME 280 • NUMBER 47 mass spectrometric analysis of nucleolar proteins suggested the presence of Mi-2␤ (CHD4) in the nucleolus (44), we investigated whether Mi-2 is associated with FLAG-MCRS1. As shown in Fig. 1B, we found that Mi-2 co-immunoprecipitated with FLAG-MCRS1. We then examined the possibility that RFP also interacts with MCRS1, as we had previously reported an association between Mi-2 and RFP (24). Immunoprecipitation experiments revealed an association between RFP and MCRS1 (Fig. 1B). To further confirm the association of MCRS1 and Mi-2, we next performed reciprocal immunoprecipitation by using anti-Mi-2 rabbit polyclonal antibody. GFP-MCRS1 was used to avoid the superimposition of the immunoglobulin band on that of MCRS1. Fig. 1C shows that GFP-MCRS1 co-immunoprecipitated with endogenous Mi-2.

Ribosomal Gene Regulation by MCRS1, Mi-2␤, and RFP
To characterize proteins that are associated with endogenous MCRS1, we raised an anti-MCRS1 polyclonal antibody and confirmed its specificity (Fig. 1D). As shown in Fig. 1E, Mi-2 was associated with endogenous MCRS1 in HEK 293 cells. Nucleolar transcription factor UBF also co-immunoprecipitated with endogenous MCRS1 (Fig. 1E). However, we could not investigate the association between RFP and endogenous MCRS1, as the RFP band coincided with the anti-MCRS1 rabbit polyclonal antibody band used for immunoprecipitation.
We next performed a yeast two-hybrid assay to map the binding domains. We performed PCR on human testis and placenta cDNA libraries to obtain full-length MCRS1/MSP58 cDNA (GenBank TM accession number NM_006337). We cloned the full-length MCRS1 cDNA into the pACT2 vector and constructed pAS2-1 vectors that carried different regions of Mi-2␤ and RFP ( Fig. 2A). Yeast strain Y190 was then transformed with the pAS2-1 and pACT2 constructs, and interactions between full-length MCRS1 and Mi-2␤ or RFP fragments were assayed both by growth on selective medium lacking histidine, tryptophan, and leucine with 40 mM 3-amino-1,2,4-triazole and by ␤-galactosidase activity. As shown in Fig. 2B, full-length MCRS1 was specifically associated with the ATPase/helicase region of Mi-2␤. Deletion constructs of MCRS1 ( Fig. 2A) mapped the central region (MCRS1-2) as the region that interacted both with the ATPase/helicase region and with the full length of Mi-2␤. The association between the full-length Mi-2␤ and the full-length MCRS1 was relatively weak in this assay. We speculate that transcriptional repressive activity of the full-length Mi-2␤ and steric constraint of the large GAL4 fusion protein may affect the expression of yeast histidine and ␤-galactosidase genes.
Full-length MCRS1 and its central region (MCRS-2) also interacted with both full-length RFP and its coiled-coil region (Fig. 2B). Although the RFP coiled-coil region (RFP-2) interacted with both MCRS1-2 and the carboxyl-terminal region of MCRS1 (MCRS1-3), full-length RFP interacted with only MCRS1-2. Taken together, we concluded that MCRS1 interacted with Mi-2␤ and RFP, with the central region of MCRS1 acting as the binding site for the ATPase/helicase region of Mi-2␤ and the coiled-coil region of RFP.
MCRS1, Mi-2␤, and RFP Co-localize in the Nucleolus-Although MCRS1 localizes exclusively to the nucleolus (34), endogenous Mi-2 and RFP are distributed throughout the nucleoplasm in a fine granular pattern or as variously sized dot-like structures. To explore the possibility that some of these domain-like structures represented the nucleolar distribution of Mi-2 and RFP, we transfected human colorectal adenocarcinoma SW480 cells with the pFLAG-MCRS1 expression plasmid and checked for the co-localization of RFP and Mi-2 with MCRS1 in the nucleolus. In SW480 cells, Mi-2 and RFP appeared to co-localize with MCRS1 at the nucleolus, as revealed by the superimposition of confocal images (Fig. 3A). We also examined the localization of RFP, Mi-2, and MCRS1 in HeLa cells and obtained the same results (data not shown).
We then investigated the endogenous distribution patterns of MCRS1, Mi-2, and RFP and assessed their co-localization in the nucleolus. Immunofluorescence analysis of SW480 cells using anti-RFP or anti-Mi-2 polyclonal antibodies together with Alexa 594-conjugatedanti-MCRS1 antibody clearly showed the co-localization of these proteins in nucleoli (Fig. 3B). The distribution of RFP, Mi-2␤, and MCRS1 showed a relatively large fusing dot-like structure or a circular distribution of small fusing dot-like structure in the nucleoli of SW480 cells.
Because nucleolar protein UBF co-immunoprecipitated with MCRS1 using HEK 293 cell lysates (Fig. 1D), we further investigated whether MCRS1, Mi-2, and RFP also co-localized with UBF. Fig. 4 shows that MCRS1, RFP, and MCRS1 co-localized with UBF in the nucleoli of SW480 cells. It has been shown that UBF localizes to the dense fibrillar component and at the periphery of the fibrillar centers in the nucleoli, where active ribosomal DNA transcription occurs by associating with RNA polymerase I and by inducing the chromatin remodeling (1,45,46). Co-localization of MCRS1, Mi-2, and RFP with UBF in the nucleoli suggested that these proteins may be associated with the regulation of nucleolar activity and ribosomal gene transcription.
MCRS1, Mi-2, and RFP Are Associated with Human rDNA-To investigate the recruitment of MCRS1, Mi-2, and RFP to the ribosomal DNA regions, ChIP assays were performed. Ribosomal genes are aligned as tandem repeats at nucleolar organizer regions on chromosomes 13, 14, 15, 21, and 22 (2). Each ribosomal DNA repeating unit is about 43 kilobases in length, with about 13 kilobases of 47 S ribosomal RNA transcribed from each unit (Fig. 5A). We designed two sets of primers within the promoter region, one set of primers within the 28 S ribosomal RNA coding region and two sets of primers within the 3Ј non-coding region (Fig. 5A). As shown in Fig. 5B, Mi-2 and RFP were associated with both the promoter regions and the 28 S rRNA coding region. In addi-tion, the proteins also bound to the 3Ј non-coding regions examined. These results suggested that Mi-2 and RFP bound to the entire rDNA region. The distribution profiles of Mi-2 and RFP matched the proposed distribution of UBF, an essential component of the RNA polymerase I initiation complex, which is distributed throughout the ribosomal gene repeats rather than restricted to the promoter region (47). However, MCRS1 was associated with both the promoter regions and the 28 S rRNA coding region but not with the 3Ј non-coding regions (Fig. 5C). This suggested that the co-operative role of MCRS1 with Mi-2 and RFP may be restricted to the modulation of rRNA transcription and that MCRS1 is not associated with Mi-2 and RFP in 3Ј-non-coding regions. However, because Mi-2 and RFP are associated with the entire ribosomal gene repeat, these proteins might also be involved in the modulation of chromatin structure, like UBF.
MCRS1, Mi-2␤, and RFP Activate rRNA Promoter Activity-The colocalization of MCRS1, Mi-2, and RFP with rRNA transcription factor UBF in the nucleolus together with the association of MCRS1, Mi-2, and RFP with ribosomal gene repeats strongly suggested that these proteins are involved in ribosomal gene transcription. To investigate this hypothesis, we cloned the human rRNA promoter region spanning from Ϫ483 to ϩ377 bp and constructed the pGL3-GAL4-hrDNAP-IRES-Luc luciferase vector (Fig. 6A). To minimize a luciferase gene expression driven by RNA polymerase II, the Kozak sequence of the pGL3 vector was replaced by the IRES sequence as previously described (42,48). It is known that the human rRNA promoter is not transcribed by the mouse pol I transcription machinery and vice versa (49). By taking advantage of the species specificity of pol I transcription, Ghoshal et al. (42) demonstrated the pol I-specific transcription of the luciferase reporter construct that contained the human rRNA promoter region (from Ϫ410 to ϩ314) (42). We confirmed the pol I-specific transcription of our pGL3-GAL4-hrDNAP-IRES-Luc luciferase vector by comparing the luciferase activities in human HEK 293 cells and mouse L cells. Fig. 6B shows that the transcriptional activity of the pGL3-GAL4-hrDNAP-IRES-Luc luciferase vector is markedly lower in mouse L cells Nucleoli were then stained with anti-UBF mouse monoclonal antibody followed by Alexa 594-labeled anti-mouse IgG antibody. The two images were merged digitally. NOVEMBER 25, 2005 • VOLUME 280 • NUMBER 47 than in human HEK 293 cells, demonstrating the pol I-specific transcription of the pGL3-GAL4-hrDNAP-IRES-Luc vector.

Ribosomal Gene Regulation by MCRS1, Mi-2␤, and RFP
Transcriptional activity of the rRNA promoter was assessed by the transient transfection of GAL4-DBD-fused MCRS1, Mi-2␤, or RFP constructs with pGL3-GAL4-hrDNAP-IRES-Luc luciferase plasmid into HEK 293 cells. As shown in Fig. 6C, Mi-2␤ strongly transactivated rRNA promoter activity by ϳ400%, whereas RFP and MCRS1 transactivated rRNA promoter activity by ϳ200%. We have previously shown that Mi-2␤ and RFP repressed the RNA polymerase II-driven luciferase activity by about 70% in the nucleus (24,32). Thus, our results indicated that Mi-2␤ and RFP have distinct transcriptional activities in the nucleus and the nucleolus.
To further assess the transcriptional activity of each region of MCRS1, Mi-2␤, and RFP, we constructed a series of GAL4-DBD-NoLSfused deletion constructs (Fig. 6D). The NoLS was used to ensure nucleolar localizations of the deletion constructs. As shown in Fig. 6E, both the central region of MCRS1 (i.e. the Mi-2␤ and RFP binding site) and the carboxyl-terminal region that contains the forkhead-associated domain transactivated the rRNA promoter. In contrast, the amino-terminal region of MCRS1 showed no such transactivating activity. We recently reported that the amino-terminal region of Mi-2␤ had strong transactivating activity, whereas the carboxyl-terminal region of Mi-2␤ had repressing activity in the nucleus (24). Surprisingly, assessment of transcriptional activities of Mi-2␤ deletion constructs revealed that the repressive activity of the carboxyl-terminal region was completely lost, such that all three regions examined transactivated the rRNA promoter (Fig. 6F). We speculate that the difference of associating proteins between nucleolar pol I transcription machinery and nuclear pol II transcription machinery may alter the transcriptional activity of Mi-2␤ and its carboxyl-terminal region.
We next investigated the transcriptional activities of RFP deletion constructs. Fig. 6G demonstrates that the coiled-coil region of RFP (i.e. A, schematic representation of the structure of a human rDNA-repeating unit region and the PCR primers used for the ChIP assay. B, association of Mi-2 and RFP with rDNA. HEK 293 cells were fixed with formaldehyde, and cross-linked chromatin was precleared and immunoprecipitated with anti-Mi-2 or anti-RFP rabbit polyclonal antibodies. Eluted DNA was decross-linked and amplified using specific primers. Input represents 1% of the sample for immunoprecipitation. C, association of MCRS1 with rDNA. HEK 293 cells were fixed and immunoprecipitated with anti-MCRS1 antibody. Eluted DNA was amplified as described above. the Mi-2␤ and MCRS1 binding site) transactivated the rRNA promoter, whereas the RFP domain region had weak transactivating activity. In contrast, the RING finger B-box region showed no transactivation function. These results suggested that the repressing functions of RFP and Mi-2␤ subdomains were not observed in the nucleolus and that MCRS1, Mi-2␤, and RFP are involved in the transactivation of ribosomal gene transcription.
siRNA-mediated Down-regulation of MCRS1, Mi-2␤, and RFP Inhibited rRNA Production-To determine the effect of MCRS1, Mi-2␤, and RFP on rRNA production in vivo, we treated HeLa cells with siRNA against the respective genes. Treatment of HeLa cells with MCRS1, Mi-2␤, and RFP siRNA resulted in significant reductions of the respective mRNAs and proteins compared with treatment with the control siRNA (Fig. 7A). We further confirmed that siRNAs used specifically repressed their target molecule and did not affect the expressions of other molecules such as MCRS1, Mi-2␤, RFP, and UBF (Fig. 7A).
Finally we assessed the amounts of rRNA in HeLa cells treated with the specific siRNAs. Human rRNA is transcribed as a primary 47 S precursor and is subsequently processed to generate mature 28 S, 18 S, and 5.8 S through the activities of different RNA-processing enzymes (Fig. 7B). PCR primer sets specific for 5ЈETS-18 S rRNA region and 28 S rRNA region were designed to measure the amounts of 47 S/45 S rRNA and subtotal rRNA, except for 18 S and 5.8 S, respectively (Fig. 7B).
Reverse transcription-PCR experiments demonstrated that 28 S rRNA region production was inhibited by 24 -43% when HeLa cells were treated with MCRS1, Mi-2␤, or RFP siRNAs (Fig. 7, C and E). The earlier processed 47 S/45 S rRNA production was more significantly reduced by 52-59% (Fig. 7, D and E). These results agreed with the transactivating activities of MCRS1, Mi-2␤, and RFP for the rRNA promoter as shown in Fig. 6 and indicated that MCRS1, Mi-2␤, and RFP are involved in the up-regulation of ribosomal gene transcription in vivo.

Transcriptional Function and Expression Profile of MCRS1 in the
Nucleolus-MCRS1 is a nucleolar protein that is highly expressed in microspherules within the nucleolus. The numbers of microspherules and the size and density of the dense fibrillar centers in the nucleolus are associated with increased nucleolar activity and the production of pre- ribosomal RNA and pre-rRNP particles. Although the finding that UBF, RNA polymerase I, fibrillarin, and p130 along with MCRS1 localized to microspherules suggested an association between MCRS1 and ribosomal gene transcription or ribosomal subunit maturation (34, 50 -53), the transcriptional roles of MCRS1 have not yet been elucidated.
In this report we examined the role of MCRS1/MSP58 in the transactivation of the ribosomal genes. MCRS1 was associated and co-localized with RFP and chromatin remodeling protein Mi-2␤ in the nucleolus. MCRS1, Mi-2␤, and RFP up-regulated ribosomal gene promoter activity and co-localized with UBF, an essential component of the RNA polymerase I initiation complex and a transactivator of RNA polymerase I-mediated transcription. In addition, down-regulation of MCRS1, Mi-2␤, and RFP by siRNA treatment inhibited the rRNA production. These findings support the view that MCRS1, Mi-2␤, and RFP are involved in RNA polymerase I-mediated transcriptional activation.
Transactivating Role of Mi-2␤ and RFP in the Nucleolus-Mi-2␤ is a major component of the well characterized NuRD complex. Mi-2␤ forms a complex with histone deacetylase HDAC1, methyl CpG-binding protein MBD2/3, metastasis-associated protein MTA 1/2/3, and histone-binding protein RbAp 48/46 and is involved in transcriptional repression in the nucleus. In a previous report we found that RFP acted as a transcriptional co-repressor that was associated with Polycomb-group protein, Enhancer of Polycomb 1, and Mi-2␤ in the nucleus (24,32).
We first speculated that MCRS1, Mi-2␤, and RFP formed a repressive complex in the nucleolus, as proposed in the nucleus. To our surprise, a transcriptional assay using a ribosomal gene promoter revealed that Mi-2␤ and RFP exhibited transactivating activities in the nucleolus. In addition, the strong repressive activities of both the Mi-2␤ carboxylterminal region and the RFP coiled-coil region were lost, with significant transactivation of the ribosomal gene promoter observed. Riboso- mal RNA is transcribed via RNA polymerase I machinery, and messenger RNA is transcribed via RNA polymerase II machinery. Likewise, the processing of transcribed RNA is also different between the nucleus and the nucleolus. Our findings indicated that Mi-2␤ and RFP had distinct transcriptional activities between the nucleus and the nucleolus and suggested that differences in associating proteins may explain these distinct transcriptional activities.
Role of Mi-2␤ and RFP in Chromatin Structure Modulation within Ribosomal Gene Repeats-ChIP assays revealed that Mi-2␤ and RFP were associated with the entire rDNA region, including the promoter region, the 47 S rRNA coding region, and the 3Ј-non-coding region. Human cells contain about 400 ribosomal genes in tandem repeats at nucleolus organizer regions encoded on five pairs of chromosomes 13, 14, 15, 21, and 22. Previous studies have indicated that only 30 -50% of rRNA genes display an open accessible chromatin structure (3,4).
Recent evidence has proposed that histone modification and chromatin remodeling proteins are highly associated with the formation of accessible or silent chromatin structures within the nucleolus organizer regions in the nucleolus in the same way as in the nucleus. Demethylation of histone H3 lysine 9 is associated with ribosomal gene silencing, whereas acetylation of H3 lysine 9 and additional histone modification are involved in transactivation (54). The chromatin remodeling complex NoRC, containing TIP5 and SNF2h, represses RNA polymerase I transcription by recruiting histone deacetylase complex containing HDAC1, Sin3A, TTF-I (transcription terminator factor), and RbAp46 (11,13).
Although UBF was originally characterized as a transactivator of RNA polymerase I by associating with the TATA box-binding proteincontaining complex SL1, recent studies have revealed that UBF is distributed throughout ribosomal DNA and activates ribosomal gene transcription through the induction of the chromatin decondensation (47,55). The distribution of Mi-2␤ and RFP throughout the entire ribosomal gene region and their co-localization with UBF within the nucleolus suggest that Mi-2␤ and RFP may be involved both in the transactivation of ribosomal gene transcription and in the formation of active chromatin structures that induce chromatin decondensation in the nucleolus.
Activation of Ribosomal Gene Transcription-The transcription of ribosomal gene repeats is thought to be controlled by the coordination of activating and repressing chromatin remodeling proteins. Although the chromatin remodeling complex NoRC is a well recognized repressive complex (12)(13)(14), the chromatin remodeling protein that is involved in transactivation remains largely unknown. Although the SWI/SNF chromatin remodeling complex has been characterized as a transactivator in the nucleus, their components have not been associated with actively transcribed regions in the nucleolus (55).
Based on the results of our study, it is possible that Mi-2␤ acts as a chromatin remodeling protein involved in ribosomal gene transactivation. Although Mi-2␤ is a major component of the NuRD complex involved in transcriptional repression in the nucleus, we have previously shown that Mi-2␤ contains both amino-terminal transactivating and carboxyl-terminal repressive regions (24). In addition, a recent report has revealed that Mi-2␤ played a direct transactivating role in CD4 expression in T cells by associating with the E box-binding protein HEB and histone acetyltransferase p300 (27). These findings suggest that another complex other than NuRD might be involved in the formation of active transcriptional loci in ribosomal gene repeats. UBF and histone acetyltransferase Tip60 are reported to reside at sites of active ribosomal DNA transcription (16,45), and our immunofluorescence experiments showed the co-localization of MCRS1, Mi-2␤, RFP, and UBF in the nucleolus. Thus, it is possible that Mi-2␤ can facilitate the formation of an active chromatin structure in the nucleolus by remodeling chromatin and associating with histone acetyltransferases such as Tip60 or p300.
Our study investigated the transcriptional function of nucleolar protein MCRS1/MSP58 and its interaction with Mi-2␤ and RFP. We identified the Mi-2␤ as a candidate molecule for the chromatin remodeling protein that is involved in the formation of open chromatin structures that facilitate active rRNA gene transcription. Our results contribute to the understanding of active chromatin structure formation at ribosomal gene repeats in the nucleolus.


s2fieldsofstudy: Biology Chemistry

text:
Probing a rate-limiting step by mutational perturbation of AdoMet binding in the HhaI methyltransferase

DNA methylation plays important roles via regulation of numerous cellular mechanisms in diverse organisms, including humans. The paradigm bacterial methyltransferase (MTase) HhaI (M.HhaI) catalyzes the transfer of a methyl group from the cofactor S-adenosyl-l-methionine (AdoMet) onto the target cytosine in DNA, yielding 5-methylcytosine and S-adenosyl-l-homocysteine (AdoHcy). The turnover rate (kcat) of M.HhaI, and the other two cytosine-5 MTases examined, is limited by a step subsequent to methyl transfer; however, no such step has so far been identified. To elucidate the role of cofactor interactions during catalysis, eight mutants of Trp41, which is located in the cofactor binding pocket, were constructed and characterized. The mutants show full proficiency in DNA binding and base-flipping, and little variation is observed in the apparent methyl transfer rate kchem as determined by rapid-quench experiments using immobilized fluorescent-labeled DNA. However, the Trp41 replacements with short side chains substantially perturb cofactor binding (100-fold higher KDAdoMet and KMAdoMet) leading to a faster turnover of the enzyme (10-fold higher kcat). Our analysis indicates that the rate-limiting breakdown of a long-lived ternary product complex is initiated by the dissociation of AdoHcy or the opening of the catalytic loop in the enzyme.

INTRODUCTION
DNA methylation plays important roles in the regulation of numerous cellular mechanisms in diverse organisms ranging from bacteriophages to humans. DNA cytosine-5 MTases (C5-MTases) transfer a methyl group from the cofactor S-adenosyl-L-methionine (AdoMet) onto the carbon-5 of cytosine in DNA. In higher organisms, DNA methylation is important for transcriptional regulation, genomic imprinting and silencing of retroviruses (1). Bacterial MTases are usually smaller than those from eukaryotes, and are often found as components of restriction-modification systems (2). Both classes of enzymes share a set of 10 conserved sequence motifs, some of which can be identified in most other Ado-Met-dependent enzymes (3). Given the high-structural homology of C5-MTases (2), it is likely that these enzymes, including the eukaryotic paralogs, also share many mechanistic features among themselves.
The paradigm model for structural and mechanistic studies is the HhaI MTase (M.HhaI), a component of a type II restriction-modification system from Haemophilus haemolyticus. M.HhaI recognizes the tetranucleotide sequence GCGC and methylates the inner cytosine residue (underlined). Crystallographic studies of M.HhaI revealed that DNA binds in a cleft of the bilobal protein, leading to dramatic conformational changes in both the DNA and the enzyme itself (4,5). An MTase-mediated rotation of the target nucleotide out of the DNA helix (base-flipping) serves to deliver the base into a concave catalytic site in the enzyme. Base-flipping is accompanied by a massive movement of the catalytic loop (residues 81-99) towards the DNA, which locks the flipped-out base for methylation (see Figure 1A). The enzymatic catalysis involves the transient formation of a Michael adduct between the catalytic cysteine (Cys81 in M.HhaI) and C6 of the cytosine, permitting a direct S N 2 transfer of the methyl group onto C5. The reaction cycle is completed by b-elimination at the C5-C6 bond of the resulting dihydro-cytosine intermediate, the reversal of the conformational changes in both protein and DNA, and the dissociation of products, AdoHcy and methylated DNA. Pre-steady and steady-state kinetic analyses of M.HhaI demonstrated that the rate of covalent methyl transfer, k chem = 0.26 s À1 , is considerably faster than the enzymatic turnover, k cat = 0.04 s À1 (6,7). Interestingly, a pre-steady burst was also observed for two other C5-MTases, the bacterial MTase MspI (8) and the mouse Dnmt1 MTase (9), indicating that, in all cases examined, a step subsequent to covalent methylation determines the rate of the reaction cycle.
However, the exact molecular event responsible for the slow turnover of the C5-MTases has not been identified.
The cofactor AdoMet binds to the larger catalytic domain of M.HhaI in a pocket facing the active site cleft (7,10,11). Residues from conserved motifs I-V contribute to the binding pocket. The outer wall of the pocket is formed by the protruding Trp41 (motif II), whose indole ring intimately stacks to the adenine ring of the bound cofactor ( Figure 1B and C). Studies with synthetic AdoHcy analogs suggested that the molecular anchor for cofactor binding is its adenosyl moiety (12). Recently, conserved residues Phe18 (conserved motif I), Trp41 (motif II), Asp60 (motif III) and Leu100 (motif V) involved in cofactor binding have been subjected to site-directed mutagenesis and biochemical studies (13,14). However, only conservative replacements were analyzed in some detail, and the functional significance of these residues could not be fully evaluated. In addition to its primary function as the methyl group donor, the cofactor AdoMet (and its product AdoHcy) was shown to play important roles in stabilizing the locked conformation of the reaction complexes (6,7,(15)(16)(17).
To assess the functional importance of cofactor interactions during catalysis, Trp41 in M.HhaI was replaced by residues of a different size. The effects of individual replacements on methylation activity, catalytic parameters, DNA binding and base-flipping were studied using a protection assay, fluorescence spectroscopy and steady-state kinetic analysis. Rapidquench single-turnover kinetic experiments were performed using a newly developed procedure that utilizes immobilized fluorescent-labeled DNA substrates. We found that the Trp41 mutations, which selectively perturb binding of AdoMet and AdoHcy, facilitate enzymatic turnover. Our analyses suggest that the breakdown of the ternary product complex could be initiated by the dissociation of AdoHcy or the opening of the catalytic loop in the enzyme, which for the first time identifies the rate-limiting events in the catalytic cycle of a DNA cytosine-5 MTase.

Oligonucleotides
Oligonucleotides were obtained from MWG-Biotech AG (HPSF grade) or Fermentas (gel-purified). Oligonucleotides for electrophoretic binding experiments were 5 0 -32 P-labeled using a DNA labeling kit (Fermentas). DNA duplexes for kinetic and biochemical studies were produced by annealing appropriate oligonucleotides (Table 1) as described previously (18).

Protein expression and purification
M.HhaI and its variants were expressed in Escherichia coli ER1727 cells containing the pHH553 plasmid (19). MTases were selectively enriched exploiting a high salt (0.4 M NaCl) back-extraction from the cell debris. Following extensive dialysis to remove bound AdoMet, MTases were purified by passing through a pre-column of Q-Sepharose followed by column chromatography on S-Sepharose (18). All proteins were purified to >95% purity as judged on Coomassie-stained polyacrylamide gels (data not shown). Protein concentrations were estimated using a Coomassie G-250 assay with BSA as standard and further refined by active site titration with a 37mer fluorescent duplex GPGC/GMGC (see below). The molecular mass of each mutant was verified by ESI-mass spectrometry (ESI-MS).

Electrophoretic gel-mobility shift assays
Dissociation constants were estimated by titrating 10 or 50 pM 5 0 -32 P-labeled hemimethylated 37mer DNA duplex GCGC/ GMGC (see Table 1) with increasing protein concentrations (0.01-100 nM). Ternary complex formation was monitored with 1 or 5 pM DNA and 0.2 pM-10 nM MTase in the presence of 500 mM AdoHcy. Samples of 20 ml were preincubated in the reaction buffer (10 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 50 mM NaCl and 2 mM 2-mercaptoethanol) containing 0.2 mg/ml of BSA and 10% glycerol for 30 min at room temperature. Aliquots were loaded onto an 8% polyacrylamide gel and fractionated by electrophoresis in 45 mM Tris-borate, pH 8.3 and 1 mM EDTA at 10 V/cm for 1 h. Dried gels were analyzed using a Cyclone PhosphorImager (Packard Instruments). Bound and free bands were quantified with Optiquant software and data were fit to the full quadratic equation for single-site binding using Grafit software (20).

Fluorescence spectroscopy
Measurements of 2-aminopurine fluorescence were performed at 25 C on a Perkin-Elmer LS-50B luminescence spectrometer equipped with a Xe lamp and a 4-mm rectangular quartz cell as described previously (18). The 200 nM 37mer GPGC/GMGC duplex (Table 1) was titrated by an incremental addition of 2 mM M.HhaI in the reaction buffer. Fluorescence intensity was recorded at l Ex = 320 nm and l Em = 370 nm with excitation and emission bandwidths of 2.5 and 5 nm, respectively. Emission spectra (340-420 nm) were recorded at an excitation wavelength (l Ex ) of 320 nm, and excitation spectra (260-350 nm) were recorded at an emission wavelength (l Em ) of 370 nm. At least three scans were averaged for each spectrum. Control scans were collected under identical conditions except that the GAGC/GMGC duplex was used instead of fluorescent DNA. The corrected spectra were obtained by subtracting the controls to eliminate contributions from the protein and other components (21).

Steady-state kinetics
Methylation reactions were carried out in the methylation buffer (50 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 50 mM NaCl, 2 mM 2-mercaptoethanol and 0.2 mg/ml of BSA) at 37 C for 10 min. K AdoMet M measurements were performed with constant (50 or 500 pM) MTase and poly(dG-dC) DNA (500 nM) and varying [methyl-3 H]AdoMet concentration from 2 nM to 200 mM. Reactions were stopped by adding HCl to 0.5 N concentration. Duplicate samples were spotted onto DE-81 filters and processed as described previously (7). The quenching of 3 H counts on DE-81 filters was estimated with poly (dG-5-[ 3 H]methyldC) and the obtained quenching factor of 1.6 was used to correct the counter readings. Data were analyzed by non-linear regression fitting to a Michaelis-Menten

Hydrogen exchange assay
Reactions containing 12 mM of both MTase and unlabeled 24mer GCGC duplex (Table 1) were incubated for 30 min at 22 C in the reaction buffer containing 80% D 2 O. Samples were then treated with Nuclease P1 and alkaline phosphatase and analyzed on an integrated HPLC/ESI-MS Hewlett-Packard 1100 series system. A sample of 200-300 pmol of total nucleosides was injected into a Discovery C18 column and was eluted with 20 mM ammonium formate, pH 3.5, for 5 min followed by a linear gradient to 55% methanol over 15 min at a flow rate of 0.3 ml/min at 45 C. The dCyd peak (elution time 3.4 -0.1 min) was analyzed in a mass detector operating at a capillary voltage of 5000 V. Spectra were collected in the positive ion mode and mass range of 108-600 m/z to monitor the presence of Cyt-Na+ (134 m.u.) and dCyd-Na+ (250 m.u.) ions.

Equilibrium dialysis
Fifty microliter of enzyme solution (30-200 mM) in a Slide-A-Lyzer 1 Mini Dialysis Unit (10 000 MWCO) was equilibrated against 1 ml of 2-50 mM [methyl-3 H]-AdoMet (4-5 concentration points for each mutant) in the reaction buffer containing 200 mM NaCl, by gently mixing at 8 C for 48 h. Duplicate aliquots from both chambers were withdrawn and radioactivity was determined in 2 ml of Rotiszint Eco Plus scintillation cocktail (Roth) using a Beckman LS 1801 liquid scintillation spectrometer. Protein concentration in each equilibrated sample was verified using a Coomassie G-250 assay with BSA as standard. The amount of bound AdoMet was calculated from the difference of radioactivity measured in protein-AdoMet and AdoMet chambers, and the data were fitted to a single-site ligand binding model using DynaFit (22).

Pre-steady-state kinetics
Single-turnover reactions were performed at 37 C in the reaction buffer containing 0.2 mg/ml of BSA. Porvair Sciences) for JOE fluorescence measurements. Fluorescence was quantified with a Perkin-Elmer LS-50B luminescence spectrometer equipped with a well plate reader accessory. Three readings were taken at an excitation wavelength of 526 nm and emission wavelength of 550 nm and averaged for each well. Reaction progress was analyzed by fitting data to a single exponential equation using Grafit (20) or DynaFit (22).

Analysis of a rate-limiting step
The K AdoMet M $k cat dependence was analyzed by fitting experimental values with the Grafit program (20) to the mechanisms described in Figure 5A and B according to the following equations: Experimental sets of k cat and K M from each mutant where used to refine global values for k chem , k unlock and f.

Construction and in vivo characterization of Trp41 mutants
As noted above, Trp41 forms the outer wall of the cofactor pocket and stacks against the adenine ring of the bound Ado-Met ( Figure 1C). Substitutions at residue 41 were chosen to vary the size of the side chain up to its complete elimination. Site-directed mutagenesis with degenerate primers was used to replace Trp41 with the following amino acid residues: Phe, Leu, Ile, Val, Ala and Gly. Sequencing of the clones produced additionally revealed the Pro and Arg variants, which were also included in further experiments. The functional ability of the mutant MTases to modify the target GCGC sites in vivo was assessed by the resistance of plasmid DNA isolated from the corresponding clones against cleavage with a methylationsensitive restriction endonuclease (21). All eight substitutions at position-41 lead to protection levels comparable to that of the WT MTase (data not shown). The mutants and WT proteins were overexpressed and purified to near homogeneity as described previously (18).

DNA binding and base-flipping activity of Trp41 mutants
We have determined the binding constants of the Trp41 mutant MTases to a 37mer duplex substrate containing a unique hemimethylated GCGC site using electrophoretic gel-mobility shift titration experiments. All but one of the mutants bound the DNA substrate with <2-fold difference in affinity from that of the WT M.HhaI ( Table 2). The W41R mutant binds several fold better to DNA than the WT enzyme most probably due to the positive charge of its side chain.
Similar experiments were performed in the presence of the cofactor AdoHcy to measure MTase-DNA interaction in the ternary complex. Previous studies of the WT enzyme showed that the addition of AdoHcy leads to the formation of a stable dead-end ternary M.HhaI-DNA-AdoHcy complex. All mutant MTases bound DNA in the presence of 500 mM AdoHcy to the same extent as the WT enzyme ( Table 2). The unaltered ability of these mutants to bind DNA in the binary and ternary complexes indicates that the Trp residue is of little importance for MTase-DNA interactions.
The capacity to flip out the target base upon interaction with DNA was examined in two ways. First, titrations with the hemimethylated duplex containing 2-aminopurine at the target site were performed. As expected, the addition of saturating amounts of the MTases results in a strong increase of 2-aminopurine fluorescence intensity (18). Fluorescence titration amplitudes and corrected excitation and emission spectra were identical to those of the WT enzyme (data not shown), suggesting that the mutations affect neither the efficiency of flipping nor the environment of the flipped 2-aminopurine base. Second, we also examined the mutants using a mechanism-based property of M.HhaI to catalyze the exchange of the hydrogen-5 atom on the target cytosine for protons of water in the absence of cofactor (23). The exchange reaction requires the formation of a transient covalent bond to the cytosine ring, which is only possible when the target cytosine is flipped out and bound in the catalytic site of the enzyme. The incorporation of D from deuterated solvent into the target cytosines in a 24mer unmethylated GCGC duplex (see Table 1) was monitored using mass-spectrometric analysis. The exchange reaction was $90% complete at the 30 min time point with the WT enzyme and the mutants (data not shown), indicating that their cytosine-flipping rates differ not more than 8-fold. Our observations thus clearly indicate that the mutant MTases are proficient in base-flipping.

AdoMet binding
Previously, M.HhaI-cofactor interactions were studied using intrinsic Trp41 fluorescence (7), but for obvious reasons this approach was not possible for the Trp41 mutants. We therefore examined the stability of binary complexes for the mutant MTases using an equilibrium dialysis technique. Binding equilibria at several concentration points of labeled [methyl-3 H] AdoMet for each mutant were determined and K D values derived by fitting data to a single-site binding mechanism. The derived K D values for the WT (4.4 mM) and W41I mutant (14 mM) are in good agreement with those determined previously by fluorescent spectroscopy (6,7,13) or isothermal calorimetry (13,14). Overall, K AdoMet D for the mutant MTases were higher than those of the WT protein by 1-2 orders of magnitude (four orders of magnitude for W41P) and increased in the order: WT < W41I < W41F, W41R, W41V < W41L, W41A < W41G ( W41P ( Figure 3A). Consistent with our structural prediction, replacing Trp41 with smaller residues reduces the affinity of M.HhaI for the cofactor AdoMet.

Steady-state kinetic analysis
To better understand the role of Trp41 in cofactor interactions, steady-state kinetic analysis was performed to determine K M  for AdoMet and the multiple turnover rates (k cat ) for the mutant enzymes. Measurements were carried out at constant saturating poly(dG-dC) and varied [methyl-3 H]AdoMet concentrations as described previously, and maintaining substrate consumption <10 and 3% for DNA and the cofactor, respectively ( Figure 2A). Both kinetic parameters measured for the WT enzyme (K AdoMet M = 35 nM; k cat = 0.02 s À1 ) are in excellent agreement with those reported previously (19). K AdoMet M values for the mutant MTases were significantly higher and showed a very wide variation ( Figure 3A). The K AdoMet M was 3-to 50-fold higher for the mutant MTases with fairly large residues (W41I, W41F, W41R, W41V, W41L), but the variants with small side chains (W41A, W41G) showed even higher increases (60-and 150-fold, respectively), indicating a dramatic decrease in the affinity for cofactor as the side chain is shortened. Replacement of Trp41 with a structurally constrained proline leads to the largest change in K M value for AdoMet (>1000-fold). Limitations in accessible concentrations of radioactive AdoMet precluded the measurement of high end points, and thus K M and k cat were derived by fitting the lower half of the curve (Figure 2A).
Unexpectedly, the multiple turnover k cat values for the mutant MTases proved to be higher when compared with the WT enzyme ( Figure 3B). Replacements of Trp41 with large hydrophobic homologs (Ile, Phe, Arg, Val, Leu) gave MTases that were 1.5-to 5-fold faster than WT protein; however, the W41A and W41G mutants exhibited 7-and 10-fold higher multiple turnover rate. The observed trend points to the importance of cofactor interactions during catalysis.

Pre-steady-state kinetic analysis
In light of the higher turnover rate of some of the mutants, we examined if the rate of the methyl transfer step, k chem , is affected by the Trp41 replacements. To avoid high experimental concentrations of radioactively labeled AdoMet (because of the high K M values of the mutants), singleturnover kinetic measurements were performed under saturating concentrations of protein and unlabeled AdoMet, using a newly developed procedure. The procedure employs an unmethylated 35/34mer duplex (biotin-GCGC-JOE, see Table 1) that contains a biotin anchor on the 5 0 end and a fluorescence label on the 3 0 end. The methylation was analyzed by monitoring the release of fluorescent label from streptavidin-immobilized DNA after its digestion with the R.Hin6I endonuclease, which only cleaves unmethylated GCGC sites. The fluorescence signal followed single exponential kinetics to reveal the methylation rate constant k chem ( Figure 2B). The measured k chem = 0.17 s À1 for WT M.HhaI with unmethylated DNA was similar to previously reported values obtained with hemimethylated DNA [0.26 s À1 , (6,7)]. The Trp41 mutants showed uniform methyl transfer rates within error (0.2-0.4 s À1 ) ( Figure 3B). This observation is consistent with the high proficiency of the mutants in DNA binding and base-flipping noted above. Although the variations of the side chain at residue-41 alter the stability of the cofactor-MTase complexes, they do not perturb the conformation of the bound cofactor in the active site pocket.

Role of Trp41 in cofactor binding
Trp41 is located on the surface of the protein and its side chain makes the bulk of the outer wall of the cofactor pocket. The adenine ring of bound AdoMet (or AdoHcy) stacks against the indole ring of Trp41 (see Figure 1C). Therefore, it is not surprising that the mutations of Trp41 affect the cofactor binding affinity both in the binary (K D ) and ternary (K M ) complexes ( Figure 3A). In fact, both affinities decrease in concert in the order: Trp > Ile > Phe, Val > Arg, Leu, Ala > Gly ) Pro. This finding suggests that the same elementary step (cofactor binding) is probed by the mutations. Trp is the largest side chain and renders the best binding for the cofactor (Figure 3A), including the highest efficiency (k cat /K M ) of catalysis in M.HhaI. However, other bulky residues can substitute for Trp41 and still permit efficient catalysis. From a structural viewpoint, the ability of the side chain to support the adenine ring of the bound cofactor may be the most important aspect of the residue occupying position-41. Therefore, branching early in the side chain (at C b as in Ile, Phe, Val) appears more important than the overall bulk (Leu, Arg) of the side chain ( Figure 3A). Indeed, phylogenetic analysis of DNA C5-MTases (Figure 4) indicates that a bulky hydrophobic residue is usually found at this position, but Ile, Trp, Val and Tyr appear most often, whereas Met and Leu appear at a lower frequency. Clearly, the occurrence of Trp with its face-to-face stacking to the adenine ring of the cofactor appears to be a peculiarity of the M.HhaI system that adds efficiency, but is not essential. The most severe kinetic perturbations of the enzyme are observed in the proline mutant ( Figure 3A). Reference to available structures (5,11) suggests a significant steric repulsion arising between C g , C d of the structurally constrained side chain of the proline and the N3, C2 atoms of the adenine ring of the bound cofactor. This unfavorable interaction apparently leads to a more dramatic effect than a mere shortening or removal of the outer wall of the pocket. Consistent with this, we found no occurrences of Pro at this position of C5-MTases.
Calorimetric analyses showed that the binding of cofactor to M.HhaI is exothermic (DDG = À6.7 and À7.8 kcal/mol for AdoMet and AdoHcy, respectively) and is driven by a favorable enthalpic change (14). Solution binding studies with truncated AdoHcy analogs showed that the thermodynamic contribution of the amino acid side chain is in the order of 2.3-2.6 kcal/mol (12). Interestingly, the removal of the outer wall of the pocket (in W41G) leads to a decline in binding of a similar magnitude ($100-fold; Figure 3A), indicating that the interactions of the adenine ring are worth 2.5-3 kcal/mol Although these fractional contributions perhaps are not fully additive, it appears that each of the three parts (the amino acid part, the ribose ring and the adenine base; Figure 1C) may have comparable thermodynamic weights for cofactor recognition. It comes as no surprise that the adenosyl moiety alone (adenine + ribose) serves as a molecular anchor for cofactor binding (12), and, therefore, AdoMet analogs, in which the methylsulfonium center is replaced by another reactive (e.g. azaridine) group, can be efficiently utilized by MTases (24).
On the other hand, we find that the Trp41 replacements in M.HhaI lead to minor effects on DNA binding [K DNA bin ð Þ D and K DNA ter ð Þ D , Table 2] (except for the positively charged Arg), base-flipping or the catalytic methyl transfer ( Figure 3B, k chem ). These observations are consistent with the remote position of Trp41 with respect to the bound DNA and the flippedout cytosine (Figure 1). The Trp mutations thus selectively perturb (weaken) the interactions of AdoMet and AdoHcy with the protein, and thus can be used as valuable probes for elucidating the role of the cofactor in the catalytic mechanism.

Rate-limiting step
The enzymatic cycle of HhaI MTase involves a series of molecular events, such as binding of substrates, dramatic conformational rearrangements in both the DNA (base-flipping) and protein (closure of the catalytic loop), covalent transformations in the catalytic site of enzyme (transient covalent activation of the cytosine and methyl transfer from bound AdoMet), followed by a breakdown of the ternary complex to the release of products and the enzyme. Steady-state (15) and pre-steadystate (6,7) kinetic analyses demonstrated that a step following the covalent methyl transfer determines the rate of catalytic turnover k cat . Structural evidence indicates that the methyl transfer occurs in the tightly closed covalent ternary complex, resulting in the ternary product E L -mD-AdoHcy ( Figure 5A). In all likelihood, the ternary product complex is the most stable intermediate on the downhill pathway dominating the scene at a steady state with a half-life of $30 s. The X-ray structures of the ternary product complexes involving AdoHcy and covalently bound (5) or non-covalently bound (25) methylated DNA show intimate contacts between the flipped-out methylated cytosine, the closed catalytic loop in the enzyme and the AdoHcy molecule. It should be noted that the ternary product complex is considerably less stable than the ternary dead-end complex involving AdoHcy and unmethylated or hemimethylated DNA (15), which has a half-life extending into hours (7). The key structural difference between the two complexes is the presence of a methyl group on the C5-position of the flipped-out target cytosine (25). The methyl group is bent out of the plane of the cytosine ring and is in tight contact with the sulfur atom of AdoHcy (separation of 2.9 Å ) (25). This tight steric interaction may account for a slight dislocation (>0.5 Å ) of the sulfur atom towards the exterior, as evident (data not shown) from comparing the corresponding structures (1MHT or 4MHT against 3MHT or 5MHT). The latter two structures contain cytosine in the catalytic site, and therefore, no steric repulsion is exerted towards the sulfur atom, which in turn may adopt a more favorable conformation.
The breakdown of the ternary product will determine the rate of dissociation of the enzyme and thus the catalytic turnover. Structural considerations above suggest that the breakdown of the closed complex can be initiated by the dissociation of AdoHcy via a separate channel (i) yielding a short-lived closed binary E L -mD complex ( Figure 5A). Owing to the dynamic nature of binary M.HhaI-DNA complexes (15,17), it is obvious that the opening of the catalytic loop (ii), departure (flip-back) of the target base from the active site (iii) and dissociation of the DNA (iv) would be fast. Alternatively, the opening of the catalytic loop (ii) would greatly facilitate the release of AdoHcy (i) from the cofactor pocket (see Figure 1B) and subsequent fast decay of the complex (iii and iv) ( Figure 5B). Evidence in favor of step (i) being rate-limiting comes from our findings that the enhanced mobility of cofactor in the W41 mutants leads to a faster turnover (higher k cat ) of the enzyme. Since the rate of the covalent methyl transfer k chem is not affected by the mutations, the increase in k cat must derive from a faster breakdown of the ternary product complex. A log plot of K AdoMet M versus k cat is hyperbolic where k cat approaches k chem as the mobility of cofactor increases ( Figure 5C). This means that k chem becomes the rate-limiting step in the latter mutants, and that all subsequent steps are faster. This, in turn, implies that the rate of reversal of the transient covalent bond at C6 in the 5,6dihydrocytosine adduct via elimination of H5, which occurs after methyl transfer and must precede the opening of the loop, is faster than k chem and thus can be disregarded in kinetic analyses. This idea is consistent with recent analyses by others (26). Assuming that the rate of cofactor release from the ternary complex is proportional to K M (k off $ fÁK M ), one can fit the observed values to a simple sequential kinetic mechanism shown in Figure 5A. However, the fit was not satisfactory at the lowest K M values which, interestingly, were associated with the amino acids present in nearly half of C5-MTases: Trp and Ile (see Figure 4B). The Trp and Ile variants had larger k cat values than expected (compare with broken line in Figure 5C), meaning that their turnover rates are higher than expected if the AdoHcy dissociation from the ternary product complex were rate-limiting. This discrepancy suggests that a second decay path of the ternary product complex may be operational. Structural considerations above hint that such a path is likely the opening (unlocking) of the catalytic loop that occurs independently of the cofactor release (step ii), and which is followed by fast events (i), (iii) and (iv) ( Figure 5B). This mechanism invokes an additional rate constant, k unlock . Similar analysis gives a very good fit to the experimental data ( Figure 5C, solid line), yielding k chem = 0.21 -0.02 s À1 and k unlock = 0.013 -0.02 s À1 for all the variants, and the rate of AdoHcy release for the WT enzyme k off = 0.008 -0.02 s À1 . The calculated value for k chem is in excellent agreement with that obtained experimentally. Although derived with substantial uncertainties, the rates for the two competing decay paths (k unlock and k off ) are of the same magnitude, suggesting that both routes may be operational and contribute to the turnover rate.
If the opening of the catalytic loop indeed occurs independently of cofactor release, it can perhaps be probed in a similar manner by targeted mutations in or around the catalytic loop. Alterations that destabilize the closed conformation of the loop in the ternary complex would be expected to speed up the decay of the ternary product. Two such mutations in which stabilizing contacts to the loop are removed have recently been described. The Q237L mutation in the target recognition domain of M.HhaI eliminates a H-bond to Ser87 in the closed loop (23). Indeed, the mutant has a several-fold higher k cat and substantially higher K AdoMet M . A similar effect was observed by us (data not shown) and others (26) when another stabilizing contact to the loop is removed. The 2-amino group of the outer guanine residue on the target strand, which makes a H-bond to the backbone oxygen of Ile86 (5), is not present if poly(dI-dC) instead of poly(dG-dC) is used as a substrate. As expected, this replacement leads to a faster turnover of the enzyme and the disappearance of a pre-steady-state burst. Interestingly, both the murine and human Dnmt1 MTases show an even greater acceleration of the steady-state turnover with poly(dI-dC) versus poly(dG-dC) (27,28). Since a presteady-state burst has also been observed for the mouse enzyme (9), it cannot be excluded that the faster opening of the catalytic loop is also responsible for this effect.
In aggregate, our analysis of M.HhaI indicates that the rate-limiting breakdown of the ternary product complex is initiated either by dissociation of AdoHcy or by conformational unlocking of the complex via opening of the catalytic loop. Obviously, variations in the DNA substrate, buffer and other conditions may affect the relative rates of these competing steps. The reversal of the covalent bond to C6 via b-elimination and all other subsequent steps are fast, with negligible contributions to the turnover rate. In light of the noted structural and mechanistic similarity, many of these mechanistic observations are likely to be valid for the whole family of C5-MTases, including the physiologically important eukaryotic enzymes.